Nucleic acid amplification primers for pcr-based clonality studies

ABSTRACT

The invention relates to PCR-based clonality studies for among others early diagnosis of lymphoproliferative disorders. Provided is a set of nucleic acid amplification primers comprising a forward primer, or a variant thereof, and a reverse primer, or a variant thereof, capable of amplifying a rearrangement selected from the group consisting of a VH-JH IGH rearrangement, a DH-JH IGH rearrangement, a VK-JK IGK rearrangement, a VK/intron-Kde IGK rearrangement, a Vλ-Jλ IGL rearrangement, a Vβ-Jβ TCRB rearrangement, a Dβ-Jβ TCRB rearrangement, a Vγ-Jγ TCRG rearrangement, a Vδ-Jδ TCRD rearrangement, a Dδ-Dδ TCRD rearrangement, a Dδ-Jδ TCRD rearrangement, a Vδ-Dδ TCRD rearrangement, or a translocation selected from t(11;14)(BCL1-IGH) and t(14;18)(BCL2-IGH). The primers can be used in PCR-based clonality studies for early diagnosis of lymphoproliferative disorders and detection of minimal residual disease (MRD). Also provided is a kit comprising at least one set of primers of the invention.

The present invention relates to PCR-based clonality studies for amongothers early diagnosis of lymphoproliferative disorders. In mostpatients with suspect lymphoproliferative disorders, histomorphology orcytomorphology supplemented with immunohistology or flow cytometricimmunophenotyping can discriminate between malignant and reactivelymphoproliferations. However, in 5 to 10% of cases, making thediagnosis is more complicated. The diagnosis of lymphoid malignanciescan be supported by clonality assessment based on the fact that inprinciple all cells of a malignancy have a common clonal Origin.

The majority of lymphoid malignancies belongs to the B-cell lineage (90to 95%) and only a minority belongs to the T-cell lineage (5-7%) orNK-cell lineage (<2%). Acute lymphoblastic leukemias (ALL) are of T-cellorigin in 15 to 20% of cases, but in the group of mature lymphoidleukemias and in non-Hodgkin lymphomas (NHL) T-cell malignancies arerelatively rare, except for specific subgroups such as cutaneouslymphomas (Table 1). Consequently, the vast majority of lymphoidmalignancies (>98%) contains identically (clonally) rearrangedimmunoglobulin (Ig) and/or T-cell receptor (TCR) genes and in 25 to 30%of cases also well-defined chromosome aberrations are found, all ofwhich can serve as markers for clonality.^(1, 2)

The Ig and TCR gene loci contain many different variable (V), diversity(D), and joining (J) gene segments, which are subjected to rearrangementprocesses during early lymphoid differentiation.^(3, 4) The V-D-Jrearrangements are mediated via a recombinase enzyme complex in whichthe RAG1 and RAG2 proteins play a key role by recognizing and cuttingthe DNA at the recombination signal sequences (RSS), which are locateddownstream of the V gene segments, at both sides of the D gene segments,and upstream of the J gene segments (FIG. 1). Inappropriate RSS reduceor even completely prevent rearrangement.

The rearrangement process generally starts with a D to J rearrangementfollowed by a V to D-J rearrangement in case of Ig heavy chain (IGH),TCR beta (TCRB), and TCR delta (TCRD) genes (FIG. 1) or concerns directV to J rearrangements in case of Ig kappa (IGK), Ig lambda (IGL), TCRalpha (TCRA), and TCR gamma (TCRG) genes. The sequences betweenrearranging gene segments are generally deleted in the form of acircular excision product, also called TCR excision circle (TREC) or Bcell receptor excision circle (BREC) (FIG. 1).

The Ig and TCR gene rearrangements during early lymphoid differentiationgenerally follow a hierarchical order. During B-cell differentiation:first the IGH genes rearrange, then IGK, potentially resulting in IgH/κexpression or followed by IGK deletion and IGL rearrangement,potentially followed by IgH/λ expression.⁵ This implies that virtuallyall Igλ⁺ B-cells have inonoallelic or biallelic IGK gene deletions.During T-cell differentiation: first the TCRD genes rearrange, thenTCRG, potentially resulting in TCRγδ expression or followed by furtherTCRB rearrangement and TCRD deletion with subsequent TCRA rearrangement,potentially followed by TCRαβ expression. The Ig and TCR generearrangement patterns in lymphoid malignancies generally fit with theabove-described hierarchical order, although unusual rearrangementpatterns are found as well, particularly in ALL.⁶

The many different combinations of V, D, and J gene segments representthe so-called combinatorial repertoire (Table 2), which is estimated tobe ˜2×10⁶ for Ig molecules, ˜3×10⁶ for TCRαβ molecules and ˜5×10³ forTCRγδ molecules. At the junction sites of the V, D, and J gene segments,deletion and random insertion of nucleotides occurs during therearrangement process, resulting in highly diverse junctional regions,which significantly contribute to the total repertoire of Ig and TCRmolecules, estimated to be >10¹².⁵

Mature B-lymphocytes further extend their Ig repertoire upon antigenrecognition in follicle centers via somatic hypermutation, a process,leading to affinity maturation of the Ig molecules. The somatichypermutation process focuses on the V-(D-)J exon of IGH and Ig lightchain genes and concerns single nucleotide mutations and sometimes alsoinsertions or deletions of nucleotides. Somatically-mutated Ig genes arealso found in mature B-cell malignancies of follicular orpost-follicular origin.⁷

Functionally rearranged Ig and TCR genes result in surface membraneexpression of Ig, TCRαβ, or TCRγδ molecules. Based on the concept thatonly a single type of Ig or TCR molecule is expressed by a lymphocyte orlymphocyte clone, the clonally rearranged genes of mature lymphoidmalignancies might be detectable at the protein level. Detection ofsingle Ig light chain expression (Igκ or Igλ) has for a long time beenused to discriminate between reactive (polyclonal) B-lymphocytes (normalIgκ/Igλ ratio: 0.7-2.8) versus aberrant (clonal) B-lymphocytes withIgκ/Igλ ratios of >4.0 or <0.5.⁸⁻¹⁰ In the vast majority (>90%) ofmature B-cell malignancies, single Ig light chain expression can supportthe clonal origin of the malignancy.

Also, the development of many different antibodies against variabledomains of the various TCR chains allows detection of monotypic Vβ, Vγand Vδ domains, when compared with appropriate reference values.¹¹⁻¹⁶ Inthe interpretation of monotypic Vβ results using 20 to 25 antibodiesagainst different Vβ families (Table 2), one should realize thatclinically-benign clonal TCRαβ⁺ T-cell expansions (frequently CD8⁺) areregularly found in peripheral blood (PB) of older individuals.^(13, 17)These clonal T-cell expansions in PB are however relatively. small insize: <40% of PB T-lymphocytes and <0.5×10⁶/ml PB.¹³ It is not yet clearto what extent such clinically benign T-cell clones can also be found inlymphoid tissues.

The results of monotypic Vγ and Vδ domain expression should beinterpreted with caution, because in healthy individuals a largefraction of normal polyclonal TCRγδ⁺ T-lymphocytes has been selected forVγ9-Jγ1.2 and Vδ2-Jδ1 usage.^(18, 19) Consequently, high frequencies ofVγ9⁺/Vδ2⁺ T-lymphocytes in PB should be regarded as a normal finding,unless the absolute counts are 1 to 2×10⁶/ml PB. It should be noted thatmost TCRγδ⁺ T-cell malignancies express Vδ1 or another non-Vδ2 genesegment in combination with a single Vγ domain (generally notVγ9).^(15, 20)

Detection of Igκ or Igλ restricted expression or monotypic Vβ, Vγ or Vδexpression is relatively easy in flow cytometric studies of PB and bonemarrow (BM) samples of patients with mature B-cell or T-cell leukemias.However, this appears to be more difficult in tissue samples withsuspect lymphoproliferative disorders that are intermixed with normal(reactive) lymphocytes.

In contrast to the antibody-based techniques, molecular techniques arebroadly applicable for detection of clonally rearranged Ig/TCR genes aswell as well-defined chromosome aberrations. This previously concernedSouthern blot analysis, but nowadays particularly PCR techniques areused.

Difficulties in making a final diagnosis of lymphoid malignancy occur ina proportion of cases (5 to 10%) despite extensive immunophenotyping.Therefore, additional (molecular clonality) diagnostics is needed togenerate or to confirm the final diagnosis, such as in case of:

-   -   any suspect B-cell proliferation where morphology and        immunophenotyping are not conclusive;    -   all suspect T-cell proliferations (CAUTION: T-cell rich B-NHL);    -   lymphoproliferations in immunodeficient patients or transplanted        patients;    -   evaluation of the clonal relationship between two lymphoid        malignancies in one patient or discrimination between a relapse        and a second malignancy;    -   further classification of a malignancy, e.g. via Ig/TCR gene        rearrangement patterns or particular chromosome aberrations;    -   occasionally: staging of lymphomas.

For long time, Southern blot analysis has been the gold standardtechnique for molecular clonality studies. Southern blotting is based onthe detection of non-germline (“rearranged”) DNA fragments, obtainedafter digestion with restriction enzymes. Well-chosen restrictionenzymes (resulting in fragments of 2 to 15 kb) and well-positioned DNAprobes (particularly downstream J segment probes) allow detection ofvirtually all Ig and TCR gene rearrangements as well as chromosomeaberrations involving J gene segments.²¹⁻²⁸. It should be noted thatSouthern blot analysis focuses on the rearrangement diversity of Ig/TCRgene segments and therefore takes advantage of the combinatorialrepertoire.

Optimal Southern blot results for clonality assessment can particularlybe obtained with the IGH, IGK, and TCRB genes, because these genes havean extensive combinatorial repertoire as well as a relatively simplegene structure which can be evaluated with only one or two DNAprobes.^(22, 24, 28) The IGL and TCRA genes are more complex and requiremultiple probe sets.^(25, 26, 29) Finally, the TCRG and TCRD genes havea limited combinatorial repertoire, which is less optimal fordiscrimination between monoclonality and polyclonality via Southern blotanalysis.^(20, 21)

Despite the high reliability of Southern blot analysis, it isincreasingly replaced by PCR techniques, because of several inherentdisadvantages: Southern blot analysis is time-consuming, technicallydemanding, requires 10 to 20 μg of high quality DNA, and has a limitedsensitivity of 5 to 10%.²¹

Detection of rearranged Ig/TCR genes and chromosome aberrations by PCRtechniques requires precise knowledge of the rearranged gene segments inorder to design appropriate primers at opposite sides of the junctionalregions and breakpoint fusion regions, respectively.

In routine PCR-based clonality studies, the distance between the primersshould be less than 1 kb, preferably less than 500 bp. This isparticularly important for discrimination between PCR products frommonoclonal versus polyclonal Ig/TCR gene rearrangements, which is basedon the diversity of the junctional regions (diversity in size andcomposition). So far, mainly IGH and TCRG gene rearrangements have beenused for PCR-based clonality studies, because of the limited number ofprimers needed to detect V_(H)-J_(H) and Vγ-Jγ rearrangements. p Themain advantages of PCR techniques are their speed, the low amounts ofDNA required, the possibility to use DNA of lower quality, and therelatively good sensitivity of 1 to 5%, for some types of rearrangementseven <1%. Consequently, PCR techniques allow the use of small biopsies(e.g. fine needle aspiration biopsies), or the use of formaldehyde-fixedparaffin-embedded samples, which generally results in DNA of lowerquality. Therefore also archival material might be used, if needed.

Molecular clonality studies can be highly informative, but severallimitations and pitfalls might hamper the interpretation of the resultsobtained with conventional detection methods:

1. Limited Sensitivity, Related to Normal Polyclonal Background

The detection limit varies between 1% and 10% (or even 15%), dependenton the applied technique (Southern blot analysis or PCR techniques) anddependent on the relative size of the “background” of normal(polyclonal) B- and T-lymphocytes. A limited sensitivity might hamperthe detection of small clonal cell populations with less than 5 to 10%clonal lymphoid cells.

2. Clonality is Not Equivalent to Malignancy

Detection of clonality does not always imply the presence of amalignancy. Some clinically benign proliferations have a clonal origin,such as many cases of CD8⁺ (or sometimes CD4⁺) T-lymphocytosis, benignmonoclonal gammopathies, initial phases of EBV⁺ lymphoproliferations(frequently being oligoclonal) in immunodeficient patients, and benigncutaneous T-cell proliferations, such as lymphomatoid papulosis, etc.This implies that results of molecular clonality studies should alwaysbe interpreted in the context of the clinical, morphological, andimmunophenotypic diagnosis, i.e. in close collaboration withhematologists, cytomorphologists, pathologists and immunologists.

3. Ig and TCR Gene Rearrangements are not Markers for Lineage

In contrast to the initial assumption, it is now clear for more than adecade that Ig and TCR gene rearrangements are not necessarilyrestricted to B-cell and T-cell lineages, respectively. Cross-lineageTCR gene rearrangements occur relatively frequently in immature B-cellmalignancies, particularly in precursor-B-ALL (>90% of cases),³⁰ butalso acute myeloid leukemias (AML) and mature B-cell malignancies mightcontain TCR gene rearrangements.³¹⁻³³ Albeit at a lower frequency, alsocross-lineage Ig gene rearrangement occur in T-cell malignancies andAML, mainly involving the Ig heavy chain (IGH) locus.^(33,34)

Virtually all (>98%) TCRαβ⁺ T-cell malignancies have TCRG generearrangements (generally biallelic) and many TCRγδ⁺ T-cell malignancieshave TCRB gene rearrangements, implying that the detection of TCRB orTCRG rearrangements is not indicative of T-cells of the αβ or γδ T-celllineage, respectively, either. In addition to these cross-lineagerearrangements, it has been established that several lymphoidmalignancies have unusual Ig/TCR gene rearrangement patterns. Thisinformation is available in detail for precursor-B-ALL and T-ALL, butnot yet for most other lymphoid malignancies.'

4. Pseudoclonality and Oligoclonality

The detection of a seemingly clonal or seemingly oligoclonal lymphoidcell population (pseudoclonality) is rare in Southern blot analysis,unless genes with a limited combinatorial repertoire are used, such asTCRG or TCRD. This might result in faint rearranged bands, e.g.representing Vγ9-Jγ1.2 or Vδ2-Jδ1 rearrangements derived fromantigen-selected TCRγδ⁺ T-lymphocytes. Yet, this is a well-known pitfallof Southern blot analysis and will not result in rearranged bands ofhigh density.

Pseudoclonality in PCR-based clonality studies is more difficult torecognize. The high sensitivity of PCR can cause amplification of thefew Ig or TCR gene rearrangements derived from a limited number ofB-cells or T-cells in the studied tissue sample. Particularly the fewreactive (polyclonal) T-cells in a small needle biopsy or in a B-NHLsample with high tumor load might result in (oligo)clonal PCR products.Frequently the amount of such PCR products is limited. This isparticularly seen when TCRG genes are used as PCR target. Duplicate ortriplicate PCR analyses followed by mixing of the obtained PCR productsshould help to clarify whether the seemingly clonal PCR products are infact derived from different lymphocytes.

Finally, reactive lymph nodes can show a reduced diversity of the Ig/TCRrepertoire, caused by predominance of several antigen-selected subclones(oligoclonality). Particularly lymph nodes or blood samples of patientswith an active EBV or CMV infection can show a restricted TCR repertoireor TCR gene oligoclonality. Also clinical pictures of immunosuppressionare frequently associated with restricted TCR repertoires, e.g. intransplant patients or patients with hairy cell leukemia.³⁵ Recoveryfrom transplantation and hematological remission are followed byrestoration of the polyclonal TCR repertoire.^(36, 37)

5. False-Positive Results

In Southern blot analysis, false-positive results are rare and cangenerally be prevented by checking for underdigestion and by excludingpolymorphic restriction sites.²¹

False-positive PCR results comprise a serious problem, if no adequateanalysis of the obtained PCR products is performed to discriminatebetween monoclonal or polyclonal PCR products. Such discrimination canbe achieved via single-strand conformation polymorphism (SSCP)analysis,³⁸ denaturing gradient gel electrophoresis (DGGE),³⁹heteroduplex analysis (HD),^(40, 41) or GeneScanning (GS).^(42, 43)These techniques exploit the junctional region diversity fordiscrimination between monoclonal cells with identical junctionalregions and polyclonal cells with highly diverse junctional regions.

6. False-Negative Results

False-negative results are rare in Southern blot analysis if appropriateJ gene segment probes are used. Nevertheless, some uncommonrearrangements (generally non-functional rearrangements) might bemissed, such as V-D rearrangements or deletions of the J regions. PCRanalysis of Ig and TCR genes might be hampered by false-negative resultsbecause of improper annealing of the applied PCR primers to therearranged gene segments. This improper primer annealing can be causedby two different phenomena. Firstly, precise detection of all differentV, D, and J gene segments would require many different primers (Table1), which is not feasible in practice. Consequently, family primers aredesigned, which specifically recognize most or all members of aparticular V, D, or J family. Alternatively, consensus primers are used,which are assumed to recognize virtually all V and J gene segments ofthe locus under study. Family primers and particularly consensus primersare generally optimal for a part of the relevant gene segments, but showa lower homology (70 to 80%) to other gene segments. This may eventuallylead to false-negative results, particularly in Ig/TCR genes with manydifferent gene segments. In TCRG and TCRD genes this problem is minimal,because of their limited number of different gene segments.

The second phenomenon is the occurrence of somatic hypermutations inrearranged Ig genes of follicular and post-follicular B-cellmalignancies, particularly B-cell malignancies with class-switched IGHgenes.

Sufficient knowledge and experience can prevent the first four pitfalls,because they mainly concern interpretation problems. The last twopitfalls concern technical problems, which can be solved by choosingreliable techniques for PCR product analysis and by the design of betterprimer sets.

Optimization of Southern blot analysis of Ig/TCR genes during the lastten years has resulted in the selection of reliable combinations ofrestriction enzymes (fragments between 2 and 15 kb, avoiding polymorphicrestriction sites) and probes (mainly downstream of J gene segments).Although Southern blot analysis is a solid “gold standard” technique,many laboratories have gradually replaced Southern blot analysis by PCRtechnology, because PCR is fast, requires minimal amounts ofmedium-quality DNA, and has an overall good sensitivity.

Despite the obvious advantages, replacement of Southern blot analysis byPCR techniques for reliable Ig/TCR studies is hampered by two maintechnical problems:

-   -   false negative results due to improper primer annealing;    -   difficulties in discrimination between monoclonal and polyclonal        Ig/TCR gene rearrangements.

Several individual diagnostic laboratories tried to solve the problemsof the PCR-based clonality studies, but thus far no reliablystandardized PCR protocols were obtained. In contrast, many differentprimer sets are being used, which all differ in their sensitivity andapplicability.

The present invention now provides sets of nucleic acid amplificationprimers and standardized PCR protocols for detecting essentially allrelevant Ig and TCR loci and two frequently occurring chromosomeaberrations. The primers sets according to the invention comprising aforward and a reverse primer are capable of amplifying clonalrearrangements of the Ig heavy chain genes (IGH), Ig kappa chain genes(IGK), Ig lamba chain genes (IGL), TCR beta genes (TCRB), TCR gammagenes (TCRG), and TCR delta genes (TCRD) or of amplifying chromosomaltranslocation t(11;14)(BCL1-IGH) and t(14;18)(BCL2-IGH). The primers ofthe invention allow that both complete and incomplete rearrangements aredetectable and that gene segments from different V, (D), and J familiescan be recognized.

Two techniques which can be used in a method of the invention fordiscrimination between monoclonal and polyclonal Ig/TCR generearrangements are heteroduplex analysis and GeneScanning. Heteroduplexanalysis uses double-stranded PCR products and takes advantage of thelength and composition of the junctional regions, whereas inGeneScanning single-stranded PCR products are separated in ahigh-resolution gel or polymer according to their length only (FIG. 2).

107 different, specific primers for all the relevant Ig/TCR loci as wellas for t(11;14) (BCL1-IGH) and t(14;18) (BCL2-IGH), or variants thereof,are provided (see FIGS. 3 to 11). The term “variant” refers to a primerwhich differs in 1 to 5 nucleotides, preferably 1 to 3 nucleotides, fromthe size and/or position from the nucleotide of a primer sequence shown,provided that the nucleotide sequence of said variant primer contains atmost 2 mismatches, at most 1 mismatch, most preferably no mismatcheswith the target locus. In addition, a variant primer comprises a(differentially) labeled primer, i.e. a primer having a label that canbe identified or distinguished from other labels by any means, includingthe use of an analytical instrument. Examples of differentially labeledprimers are primers provided with a fluorescent label such as a 6-FAM,HEX, TET or NED dye. Labeled primers of the invention are particularlyadvantageous for use in automated high resolution PCR fragment analysis(Gene Scanning technology) for detection of PCR products. As isexemplified below, differentially labeled primers according to theinvention allow to distinguish different PCR amplification products ofapproximately the same length (size), preferably using multi-colorGeneScanning. Of course, a variant nucleic acid amplification primer, beit a forward or a reverse (dye-labeled) primer, should not be capable offorming dimers with any other (variant) forward and/or reverse nucleicacid amplification primer that is used in an amplification reaction,since this can interfere with primer annealing to a target locus andthus with the amplification of the rearrangement or translocation ofinterest.

In one embodiment, the invention provides a nucleic acid amplificationassay, preferably a PCR assay, using at least one set of primersaccording to the invention. Said PCR assay can be, a single (monoplex)or a multiplex PCR. In a preferred embodiment, a set of primersaccording to the invention is used in a standardized multiplex PCRassay, using for example two or more forward primers, or three or fourforward primers, or variants thereof (e.g. selected from a group of“family primers”, for example from the V_(H) family primers), togetherwith one or more consensus reverse primer(s), or variant(s) thereof(e.g. a J_(H) consensus primer). The family primers of the invention aredesigned in such a way that they recognize most or all gene segments ofa particular family (see Table 2). In a specific embodiment, all 107primers are used in only 18 multiplex PCR tubes: 5 for IGH (3×V_(H)-J_(H) and 2× D_(H)-J_(H)), 2 for IGK, 1 for IGL, 3 for TCRB (2×Vβ-Jβ and 1× Dβ-Jβ), 2 for TCRG, 1 for TCRD, 3 for BCL2-IGH, and 1 forBCL1-IGH (FIGS. 3 to 11). Such an assay allows assessing clonalrearrangements and/or chromosome aberrations. Furthermore, it allowsdetection of a lymphoproliferative disorder. Multiplex PCR testing ofthe primers on about 90 Southern blot defined lymphoproliferationsshowed that in more than 95% of the samples the Southern blot and PCRresults were concordant.

In another embodiment, a method is provided for detecting arearrangement, preferably two or more rearrangements, selected from thegroup consisting of a V_(H)-J_(H) IGH rearrangement, a D_(H)-J_(H) IGHrearrangement, a V_(K)-J_(K) IGK rearrangement, a V_(K)/intron-Kde IGKrearrangement, a Vλ-Jλ IGL rearrangement, a Vβ-Jβ TCRB rearrangement, aDβ-Jβ TCRB rearrangement, a Vγ-Jγ TCRG rearrangement, a Vδ-Jδ TCRDrearrangement, a Dδ-Jδ TCRD rearrangement, a Dδ-Dδ TCRD rearrangement,and a Vδ-Dδ TCRD rearrangement, using at least one set of primersaccording to the invention. Also provided is a method for detecting at(11;14)(BCL1-IGH) translocation or a t(14;18)(BCL2-IGH) translocation,using at least one set of primers according to the invention.Furthermore, methods are provided for detecting at least one of theabove rearrangements and at least one translocation, using at least twosets of primers as provided herein.

In a further aspect, a set of nucleic acid amplification primers capableof amplifying a human gene selected from the group consisting of thehuman AF4 gene (exon 3), the human AF4 gene (exon 11), the human PLZF1gene, the human RAG1 gene and the human TBXAS1 gene is provided (seeFIG. 12). Using one or more of these five primer sets consisting of aforward primer (or a variant thereof) and a reverse primer (or a variantthereof) in a nucleic acid amplification assay of the invention, it ispossible to detect one or more “Control Gene(s)” selected from the groupconsisting of the human AF4 gene (exon 3), the human AF4 gene (exon 11),the human PLZF1 gene, the human RAG1 gene and the human TBXAS1 gene.Such a detection method is advantageously used to assess the quality(e.g. integrity and amplifiability) of a nucleic acid (DNA) sampleextracted or isolated from a biological sample, for instance DNAextracted from a paraffin-embedded sample (see Example 10).

The ability of the different primer sets of the invention to amplifyclonal rearrangements and/or chromosomal aberrations (translocations)has been tested in many different types of malignant lymphomas, amongwhich follicular lymphoma, diffuse large B-cell lymphoma, and multiplemyeloma. It was found that a set of primers is very useful for assessingclonal rearrangements and/or chromosomal translocations. It appearedthat the detection rate of clonal rearrangements using the multiplexprimer tubes according to the invention is unprecedentedly high, i.e atleast 95%.

Parallel testing of available paraffin-embedded tissues of the abovesamples revealed largely identical results, if the DNA quality of thesetissues is sufficiently high, meaning that fragments of at least 300 bpcan be amplified in a specially-designed control gene PCR.

The applicability of the developed multiplex PCR assays according to theinvention was evaluated on series of 50 to 100 cases per type oflymphoid malignancy. Following national pathology panel review, andcentral pathology panel review in case of difficulties, all includedcases were defined according to the World Health Organization (WHO)classification. The studied diagnostic categories included malignanciessuch as follicular lymphoma, mantle cell lymphoma, marginal zonelymphoma, diffuse large B-cell lymphoma, angioimmunoblastic T-celllymphoma, peripheral T-cell lymphoma, and anaplastic large celllymphoma, as well as reactive lesions. The results show a very highlevel of clonality detection, even in entities, which are known to bearsomatic hypermutations such as follicular lymphoma and diffuse largeB-cell lymphoma. Particularly the usage of the three IGH V_(H)-J_(H)tubes, supplemented with the two IGH D_(H)-J_(H) tubes and the two IGKtubes appeared to be highly efficient in the detection of clonal Ig generearrangements. This high efficiency is obtained by the complementarityof the Ig tubes as well as by the fact that D_(H)-J_(H) and IGK-Kderearrangements are not (or rarely) somatically mutated. Suchcomplementarity was also found for the TCRB and TCRG primers in case ofT-cell malignancies.

Furthermore, interesting and unexpected rearrangement patterns, such asunusual cross-lineage rearrangements, were observed. Remarkably, inabout 10% of reactive lesions clonal rearrangements were detected. Thesereactive lymphoproliferations included EBV-related lymphoproliferationsand atypical hyperplasias like Castleman's disease, as well as lesionsthat were suspicious for a B- or T-cell clone.

In a specific embodiment, a method is provided for the detection ofminimal residual disease (MRD). The term minimal residual disease (MRD)describes the situation in which, after chemotherapy for acute leukemia(AL), a morphologically normal bone marrow can still harbor a relevantamount of residual malignant cells. Detection of minimal residualdisease (MRD) is a new practical tool for a more exact measurement ofremission induction during therapy because leukemic blasts can bedetected down to 10⁻⁴-10⁻⁶. Known PCR-based MRD analysis uses clonalantigen receptor rearrangements detectable in ˜90-95% of theinvestigated patient samples. However, amplification of polyclonalproducts often leads to false-positive PCR amplicons not suitable forMRD analysis. The invention now provides a method for the detection ofidentically (clonally) rearranged Ig and TCR genes or detection ofwell-defined and frequent chromosome aberrations, such as t(11;14), andt(14;18). Thus, the rearrangements and translocations detected using aset of primers of the invention not only serve as markers for clonalityat diagnosis, but also as PCR targets for detection of MRD duringfollow-up.

In a further aspect, the invention provides a (diagnostic) kit for thedetection of at least one rearrangement selected from the groupconsisting of a V_(H)-J_(H) IGH rearrangement, a D_(H)-J_(H) IGHrearrangement, a V_(K)-J_(K) IGK rearrangement, a V_(K)/intron-Kde IGKrearrangement, a Vλ-Jλ IGL rearrangement, a Vβ-Jβ TCRB rearrangement, aDβ-Jβ TCRB rearrangement, a Vγ-Jγ TCRG rearrangement, a Vδ-Jδ TCRDrearrangement, a Dδ-Jδ TCRD rearrangement, a Dδ-Dδ TCRD rearrangement, aVδ-Dδ TCRD rearrangement and/or at least one translocation selected fromt(11;14)(BCL1-IGH) and t(14;18)(BCL2-IGH), comprising at least one setof primers according to the invention. A kit of the invention is highlysuitable for PCR-based clonality diagnostics. Optionally, such a kitalso comprises at least one set of primers capable of amplifying a human“control gene” as mentioned above. Inclusion of one, preferably at leasttwo, more preferably at least three of these control gene primer sets ina Control Tube can be helpful in estimating the quality of the DNAsample to be diagnosed, for instance DNA extracted fromparaffin-embedded tissue.

In a further aspect, the invention provides a method for rapiddiscrimination of different types of Ig/TCR gene rearrangements in thesame multiplex PCR tube. GeneScanning allows the application of multipledifferent fluorochrome-conjugated primers in a single tube. Suchdifferential labeling of primers can be used for extra discriminationbetween different types of Ig or TCR gene rearrangements.

Differential labeling of V primers generally has limited added value,but differential labeling of downstream primers can support the rapidand easy identification of the type of Ig/TCR gene rearrangement, whichis useful for PCR-based detection of minimal residual disease.^(44, 45)Labeling of J primers is not regarded to be informative for IGH(V_(H)-J_(H) or D_(H)-J_(H)), IGK (Vκ-Jκ), or IGL (Vλ-Jλ). For rapididentification of IGK-Kde rearrangements, it might be interesting todiscriminate between Vκ-Kde and intron RSS-Kde rearrangements bydifferential labeling of the Kde and intron RSS primers (see FIG. 5B).

The most informative multicolor GeneScanning can be designed for TCRgene rearrangements, facilitating the rapid recognition of the differenttypes of TCRB, TCRG, and TCRD gene rearrangements. For example,differential labeling of the Jβ1 and Jβ2 primers in TCRB tube A (seeFIG. 7B) allows easy identification of the polyclonal and monoclonalVβ-Jβ1 versus Vβ-Jβ2 rearrangements (FIG. 13A). Differential labeling ofthe Jγ1.3/2.3 and Jγ1.1/2.1 primers (FIG. 8B) results in easyidentification of the different types of TCRG gene rearrangements (FIG.13B). Differential labeling of the Jδ primers, Dδ2 primer, and Dδ3primer in the TCRD tube (FIG. 9B) results in easy identification of themost relevant TCRD gene rearrangements, such as Dδ2-Jδ, Vδ-Jδ, Dδ2-Dδ3,and Vδ2-Dδ3 rearrangements (FIG. 13C).

These multi-color multiplex PCR tubes appear to be easy and convenientin daily practise of PCR based clonality diagnotics.

LEGENDS TO THE FIGURES

FIG. 1. Sche1natic diagram of sequential rearrangement steps,transcription, and translation of the TCRB gene. In this example first aDβ2 to Jβ2.3 rearrangement occurs, followed by Vβ4 to Dβ2-Jβ2.3rearrangement, resulting in the formation of a Vβ4-Dβ2-Jβ2.3 codingjoint. The rearranged TCRB gene is transcribed into precursor mRNA,spliced into mature mRNA, and finally translated into a TCR13 proteinchain. The two extrachromosomal TCR excision circles (TRECs) that areformed during this recombination process are indicated as well; theycontain the D-J signal joint and V-D signal joint, respectively.

FIGS. 2A, 2B and 2C. Schematic diagram of heteroduplex analysis andGeneScanning of PCR products, obtained from rearranged Ig and TCR genes.FIG. 2A. Rearranged Ig and TCR genes (IGH in the example) showheterogeneous junctional regions with respect to size and nucleotidecomposition. Germline nucleotides of V, D, and J gene segments are givenin large capitals and randomly inserted nucleotides in small capitals.The junctional region heterogeneity is employed in heteroduplex analysis(size and composition) and GeneScanning (size only) to discriminatebetween products derived from monoclonal and polyclonal lymphoid cellpopulations. FIG. 2B. In heteroduplex analysis, PCR products areheat-denatured (5 min, 94° C.) and subsequently rapidly cooled (1 hour,4° C.) to induce duplex (homo- or heteroduplex) formation. In cellsamples consisting of clonal lymphoid cells, the PCR products ofrearranged IGH genes give rise to homoduplexes after denaturation andrenaturation, whereas in samples which contain polyclonal lymphoid cellpopulations the single-strand PCR fragments will mainly formheteroduplexes, which result in a background smear of slowly migratingfragments upon electrophoresis. FIG. 2C. In GeneScanningfluorochrome-labeled PCR products of rearranged IGH genes are denaturedprior to high-resolution fragment analysis of the resultingsingle-stranded fragments. Monoclonal cell samples will give rise to PCRproducts of identical size (single peak), whereas in polyclonal samplesmany different IGHPCR products will be formed, which show acharacteristic Gaussian size distribution.

FIGS. 3A, 3B, 3C, 3D and 3E. PCR analysis of IGH (V_(H)-J_(H))rearrangements. FIG. 3A. Schematic diagram of IGH gene complex onchromosome band 14q32.3 (adapted from ImMunoGeneTics database).⁴⁶ ⁴⁷Only rearrangeable non-polymorphic V_(H) gene segments are included inblue (functional V_(H)), or in gray (rearrangeable pseudogenes).Recently discovered (generally truncated) V_(H) pseudogenes are notindicated. FIG. 3B. Schematic diagram of IGH V_(H)-J_(H) rearrangementwith three sets of V_(H) primers and one J_(H) consensus primer,combined in three multiplex tubes. The relative position of the V_(H)and J_(H) primers is given according to their most 5′ nucleotideupstream(−) or downstream(+) of the involved RSS. The V_(H) gene segmentused as representative V_(H) family member for primer design isindicated in parentheses. FIG. 3C, FIG. 3D, and FIG. 3E. Heteroduplexanalysis and GeneScanning of the same polyclonal and monoclonal cellpopulations, showing the typical heteroduplex smears and homoduplexbands (left panels) and the typical polyclonal Gaussian curves andmonoclonal peaks (right panels). The approximate distribution of thepolyclonal Gaussian curves is indicated in nt.

FIGS. 4A, 4B and 4C. PCR analysis of IGH (D_(H)-J_(H)) rearrangements.FIG. A. Schematic diagram of IGH (D_(H)-J_(H)) rearrangement with sevenD_(H) family primers and one J_(H) consensus primer, divided over twotubes (IGH tubes D and E). The D_(H)7 (7-27) primer was separated fromthe other six DH primers, because the D_(H)7 and J_(H) consensus primerwill give a germline PCR product of 211 nt. The relative position of theDH and J_(H) primers is given according to their most 5′ nucleotideupstream(−) or downstream(+) of the involved RSS. The D_(H) gene segmentused as representative D_(H) family member for prim. er design isindicated in parentheses. FIG. B and FIG. C. Heteroduplex analysis (leftpanels) and GeneScanning (right panels) of the same polyclonal andmonoclonal cell populations. The approximate distribution of thepolyclonal and monoclonal peaks is indicated. The potential backgroundband/peak in tube D is indicated with an asterisk and is located outsidethe expected range of D_(H)-J_(H) rearrangements. The germlineD_(H)7-J_(H) band of tube E is also indicated with an asterisk.

FIGS. 5A, 5B, 5C and 5D. PCR analysis of IGK gene rearrangements. FIG.5A. Schematic diagram of the IGK gene complex on chromosome band 2p11.2(adapted from ImMunoGeneTics database).⁴⁶ ⁴⁷ Only rearrangeablenon-polymorphic Vκ gene segments are indicated in blue (functional Vκ)or in grey (nonfunctional Vκ). The cluster of inverted Vκ gene segments(coded with the letter D) is located ˜800 kb upstream of thenon-inverted Vκ gene segments. These upstream Vκ gene segments arepresented as a mirrored image to their corresponding non-invertedcounterparts. FIG. 5B. Schematic diagrams of Vκ-Jκ rearrangement and thetwo types of Kde rearrangements (Vk-Kde and intron RSS-Kde). Therelative position of the Vκ, JκK, Kde and intron RSS (INTR) primers isgiven according to their most 5′ nucleotide upstream(−) or downstream(+)of the involved RSS. The Vκ gene segment used as representative memberof the Vκ1, Vκ2, and Vκ3 families are indicated in parentheses. Vκ4,Vκ5, and Vκ7 are single-member Vκ families. The primers are divided overtwo tubes: tube A with Vκ and Jκ primers and tube B with Vκ, intron RSS,and Kde primers. FIG. 5C and FIG. 5D. Heteroduplex analysis andGeneScanning of the same polyclonal and monoclonal cell populations,showing the typical heteroduplex smears and homoduplex bands (leftpanels) and the typical Gaussian curves and monoclonal peaks (rightpanels). The approximate distribution of the polyclonal Gaussian curvesis indicated in nt.

FIGS. 6A, 6B and 6C. PCR analysis of IGL gene rearrangements. FIG. 6A.Schematic diagram of IGL gene complex on chromosome band 22g11.2(adapted from ImMunoGenetics database).⁴⁶ ⁴⁶ Only rearrangeablenon-polymorphic Vλ gene segments are included in blue (functional Vλ) orin grey (nonfunctional Vλ) FIG. 6B. Schematic diagram of Vλ-Jλrearrangement with two Vλ family primers and one Jλ consensus primer.Only two Vλ primers were designed for Vλ1 plus Vλ2 and for Vλ3, becausethese three Vλ families cover approximately 70% of rearrangeable Vλ genesegments and because approximately 90% of all IGL gene rearrangementsinvolve Vλ1, Vλ2, or Vλ3 gene segments.⁴⁸ Although five of the sevenJλ1, gene segments can rearrange, only a single consensus primer wasdesigned for Jλ1, Jλ2, and Jλ3, because 98% of all IGL generearrangements involve one of these three gene segments.⁴⁹ The relativeposition of the Vλ and Jλ primers is given according to their most 5′nucleotide upstream(−) or downstream(+) of the involved RSS. FIG. 6C.Heteroduplex analysis and GeneScanning of the same polyclonal andmonoclonal cell populations, showing the typical heteroduplex smears andhomoduplex bands (left panel) and the polyclonal Gaussian curves andmonoclonal peaks (right panel). The approximate position of thepolyclonal Gaussian curves is indicated in nt.

FIGS. 7A, 7B, 7C, 7D and 7E. PCR analysis of TCRB gene rearrangements.FIG. 7A. Schematic diagram of the human TCRB locus. The gene segmentdesignation is according to Arden et al.⁵⁰ with the designationaccording to Rowen et al.⁵¹ and Lefranc et al.⁴⁶ ⁴⁷ in parentheses. Thefigure is adapted from the international ImMunoGeneTics database.⁴⁶ ⁴⁷Only the rearrangeable non-polymorphic Vβ gene segments are depicted inblue (functional Vβ), in half blue/half gray (potential functional, butno protein expression found) and in grey (non-functional Vβ). FIG. 7B.Schematic diagram of Vβ-Jβ and Dβ-Jβ rearrangements. The 23 Vβ primers,13 Jβ primers and two Dj3 primers are combined in three tubes: tube Awith 23 Vβ primers and nine Jβ primers, tube B with 23 Vβ primers andfour Jβ primers, and tube C with two D13 primers and 13 Jβ primers. The23 Vβ primers and the 13 Jβprimers are aligned in order to obtaincomparably sized PCR products (see panels C and D). The Vβ primers coverapproximately 90% of all Vβ gene segments. The relative position of theVβ, Dβ, and Jβ primers is given according to their most 5′ nucleotideupstream(−) or downstream(+) of the involved RSS. FIG. 7C, FIG. 7D, andFIG. 7E. Heteroduplex analysis and GeneScanning of the same polyclonaland monoclonal cell populations, showing the typical heteroduplex smearsand homoduplex bands (left panels) and the typical polyclonal Gaussiancurves and monoclonal peaks (right panels). The approximate distributionof the polyclonal Gaussian curves is indicated in nt.

FIGS. 8A, 8B, 8C and 8D. PCR analysis of TCRG gene rearrangements. FIG.8A. Schematic diagram of the human TCRG locus on chromosome band 7p14.Only the rearrangeable Vγ gene segments are depicted in blue (functionalVγ) or in gray (non-functional Vγ). For the Jγ gene segments, bothnomenclatures are used.⁴⁵ ⁴⁷ ⁵² FIG. 8B. Schematic diagram of TCRG Vγ-Jγrearrangement with four Vγ primers and two Jγ primers, which are dividedover two tubes. The relative position of the Vγ and Jγ primers isindicated according to their most 5′ nucleotide upstream(−) ordownstream(+) of the involved RSS. FIG. 8C and FIG. 8D. Heteroduplexanalysis and GeneScanning of the same polyclonal and monoclonal cellpopulations, showing the typical heteroduplex smears and homoduplexbands (left panels) and the typical polyclonal Gaussian curves andmonoclonal peaks (right panels). The approximate distribution of thepolyclonal Gaussian curves is indicated in nt.

FIGS. 9A, 9B and 9C. PCR analysis of TCRD gene rearrangements. FIG. 9A.Schematic diagram of human TCRD locus on chromosome band 14q11.2. Thesix “classical” Vd gene segments are indicated in blue, scatteredbetween the Vα, gene segments in black. Since Vδ4, Vδ5, and Vδ6 are alsorecognized as Vα gene segments, their Va gene code is given inparenthesis. FIG. 9B. Schematic diagram of Vδ-Jδ, Dδ2-Jδ, Dδ2-Dδ3, andVδ-Dδ83 rearrangements, showing the positioning of six Vδ, four Jδ, andtwo Dδ primers, all combined in a single tube. The relative position ofthe Vδ, Dδ, and Jδ primers is indicated according to their most 5′nucleotide upstream(−) or downstream(+) of the involved RSS. FIG. 9C.Heteroduplex analysis (left panel) and GeneScanning (right panel) of thesame polyclonal and monoclonal cell populations. The polyclonal cellpopulations show a vague smear in heteroduplex analysis and a complexand broad peak pattern in GeneScanning. The monoclonal bands and peaksare clearly visible. The approximate position of the PCR products of thedifferent types of rearrangements in GeneScanning is indicated.

FIGS. 10A and 10B. Detection of BCL1-IGH rearrangements. FIG. 10A.Schematic diagram of the CCND1 gene and the BCL1 breakpoint region MTCon chromosome band 11q13 as well as the J_(H) gene segment on chromosomeband 14q32. For the primer design in the BCL1-MTC region an artificialBCL1-MTC/J_(H)4 junctional sequence was composed (as partially reportedfor JVM2⁵³): the first 50 nucleotides as reported by Willams⁵⁴werelinked to nucleotide 1-439 from MTC-sequence present at NCBI(accession-number S77049 55); the N-region “GCCC” of JVM253 was addedfollowed by nucleotide 1921-3182 representing the J_(H)4-J_(H)6 genomicregion (accession-number J00256). FIG. 10B. Agarose gel electrophoresisof a series of BCLJ-IGH PCR products from different MCL patients and thepositive control cell line JVM2. The PCR products differ is size,indicating different positions of the BCL1-MTC breakpoints. The largerbands of lower density represent PCR products that extend to the nextdownstream germline J_(H) gene segment.

FIGS. 11A, 11B, 11C and 11D. PCR detection of BCL2-IGH rearrangements.FIG. 11A. Schematic diagram of the BCL2 gene on chromosome band 18q21.The majority of the BCL2 breakpoints cluster in three regions: MER, 3′MER, and mer. Consequently, multiplex primers have been designed tocover the potential breakpoints in these three regions: two MBR primers,four 3′ MBR primers, and three mer primers. The relative position of theBCL2 primers is indicated according to their most 5′ nucleotideupstream(−) or downstream(+) to the 3′ end of BCL2 exon 3 (according toNCBI accession no. AF325194S1), except for two BCL2-mer primers; theirposition is indicated downstream of the first nucleotide of the AF275873sequence. FIG. 11B, FIG. 11C, and FIG. 11D. Agarose gel electrophoresisof PCR products from different FCL patients and several positive controlcell lines (DoHH2, K231, OZ, and SC1). Panel E and D contain the samesamples and show complementarity in positivity, illustrating that tube C(mer tube) has added value. The PCR products differ in size, related todifferent position of the BCL2 breakpoints. The larger bands of lowerdensity in the same lanes represent PCR products that extend to the nextdownstream germline J_(H) gene segment or to the next upstream BCL2primer.

FIGS. 12A and 12B. Control gene PCR for assessment of amplifiability andintegrity of DNA samples. FIG. 12A. Schematic diagram of five controlgenes exons and the five primer sets for obtaining PCR products of 600bp, 400 bp, 300 bp, 200 bp, and 100 bp. The relative position of thecontrol gene primers is given according to their most 5′ nucleotidedownstream of the 5′ splice site of the involved control gene exon. FIG.12B. Control gene PCR products of six DNA samples, separated in a 6%polyacrylamide gel. Two control samples contained high molecular weightDNA (outer lanes) and four DNA samples were obtained fromparaffin-embedded tissue samples, showing reduced amplifiability (e.g.GBS-4 50 ng versus GBS-4 500 ng) or reduced integrity of the DNA (PT-4).

FIGS. 13A, 13B and 13C. Multicolor GeneScanning for supporting the rapidand easy identification of TCR gene rearrangements. FIG. 13A. Two-coloranalysis of TCRB tube A with differential labeling of Jβ1 primers(TET-labeled; green) and Jβ2 primers (FAM labeled; blue). The top panelnicely shows the two polyclonal Jβ1 and Jβ2 rearrangement patterns (c.f.FIG. 7C), whereas the other two panels show clonal Jβ2 rearrangements.FIG. 13B. Two-color analysis of TCRG tube A with differential labelingof the Jγ1.3/2.3 primer (FAM-labeled; blue) and the Jγ1.1/2.1(TET-labeled; green). The top panel nicely shows the polyclonalrearrangement patterns (c.f. FIG. 8C), whereas the other two panels showclonal Jγ1.3/2.3 and clonal Jγ1.1/2.1 rearrangements, respectively. FIG.13C. Three-color analysis of TCRD gene rearrangements with differentiallabeling of Jδ primers (FAM-labeled; blue), Dδ2 primer (HEX-labeled;green) and Dδ3 primer (NED-labeled; black). Within the complexrearrangement patterns of the TCRD tube (FIG. 9C), the three-coloranalysis allows direct detection of Vδ-Jδ rearrangements (blue peaks),Dδ2-Jδ rearrangements (blue and green peaks, not fully comigratingbecause of differences in migration speed of the two fluochromosomes),Vδ2-Dδ3 rearrangement (black peaks), and Dδ2-Dδ3 rearrangement(comigrating green and black peaks).

MATERIALS AND METHODS

Selection of PCR Targets: Aiming for Complementarity

It was decided to aim for the availability of at least onePCR-detectable clonality target in each lymphoid malignancy. In matureB-cell malignancies this aim might be hampered by the occurrence ofsomatic hypermutations in Ig genes, which are particularly found infollicular and post-follicular B-cell malignancies. Therefore it wasdecided to include PCR targets that have some degree of complementarity.

Several rationales were used for target selection:

-   -   IGH genes: not only complete V_(H)-J_(H) rearrangements but also        incomplete D_(H)-J_(H) rearrangements were included as PCR        targets, because D_(H)-J_(H) rearrangements are probably not        affected by somatic hypermutations;    -   IGK and IGL genes: both Ig light chain genes were included as        PCR targets, because this increases the chance of finding a        PCR-detectable Ig gene rearrangement in each mature B-cell        malignancy;    -   IGK genes: not only Vκ-Jκ rearrangements were included, but also        rearrangements of the kappa deleting element (Kde), because they        occur on one or both alleles in (virtually) all Igλ⁺ B-cell        malignancies and in one third of Igκ⁺ B-cell malignancies and        because Kde rearrangements are probably not affected by somatic        hypermutation;    -   TCRB genes: both complete Vβ-Jβ and incomplete Dβ-Jβ        rearrangements, because complete and incomplete TRCB gene        rearrangements occur in all mature TCRαβ⁺ T-cell malignancies        and also in many TCRγδ⁺ T-cell malignancies;    -   TCRG genes: this classical PCR clonality target is useful in all        T-cell malignancies of the TCRγδ and the TCRαβ lineage.    -   TCRD genes: this is a potentially useful target in immature        T-cell malignancies as well as in TCRγδ⁺ T-cell malignancies;    -   TCRA gene: this gene was not included as PCR target, because of        its high degree of complexity with ˜50 V and 61 J gene segments.        Furthermore, all T-cell malignancies with TCRA gene        rearrangements contain TCRB gene rearrangements and generally        also have TCRG gene rearrangements;    -   functional gene segments: most suspect lymphoproliferations        concern mature lymphocytes, which consequently have functional        Ig or TCR gene rearrangements. Therefore PCR primer design aimed        at inclusion of (virtually) all functional Ig/TCR gene segments.    -   well-defined chromosome aberrations: t(11;14) with BCL1-IGH and        t(14;18) with BCL2-IGH were included as additional targets,        because these two aberrations are PCR-detectable at relatively        high frequencies in lymphomas i.e. in 30% of mantle cell        lymphoma (MCL) and in 60 to 70% of follicular cell lymphomas        (FCL), respectively.

Primer Design for Multiplex PCR

Precise detection of all V, D, and J gene segments in rearranged Ig andTCR genes would require many different primers (Table 2). For some genecomplexes this might be possible (e.g. TCRG and TCRD), but for otherloci in practice this is impossible because of the high number ofdifferent gene segments. To solve this problem, family primers can bedesigned, which recognize most or all gene segments of a particularfamily (Table 2). Alternatively, consensus primers can be made, whichrecognize conserved sequences that occur in many or all involved genesegments.

The design of family primers and consensus primers balances between alimited number of primers and maximal homology with all relevant genesegments. In this study, we aimed at maximal homology with all relevantgene segments (particularly functional gene segments) in order toprevent suboptimal primer annealing, which might cause false-negativeresults. Furthermore, we aimed at the design of specific family primerswithout cross-annealing to other families

In order to limit the number of PCR tubes per locus, multiplexing of PCRprimers became important for practical reasons. Consequently, specialguidelines were developed to ensure maximal possibilities for designingprimers useful in multiplex PCR tubes. For this purpose dr. W. Rychlick(Molecular Biology Insights, Cascade, Colo., USA) provided hisspecially-adapted OLIGO 6.2 software program and supported thedevelopment of the guidelines for optimal primer design.

The general guidelines for primer design were as follows:

-   -   the position of the primers should be chosen in such a way that        the size of the PCR products would preferably be <300 bp        (preferably 100 to 300 bp) in order to be able to use        paraffin-embedded material;    -   a minimal distance to the junctional region of preferably >10-15        bp should be taken into account (in order to avoid        false-negativity due to impossibility of the 3′ end of the        primer to anneal to the rearranged target because of nucleotide        deletion from the germline sequence);    -   primers preferably should not be too long (e.g. <25        nucleotides).

The following parameters were used for primer design with the OLIGO 6.2program:

-   -   search for primers should be performed with moderate stringency;    -   primer efficiency (PE) value should preferably be ˜400        (and >630, if the primer is to be used as consensus primer for        other gene segments as well);    -   the most stable 3′ dimer of upper/upper, lower/lower, or        upper/lower primers should not exceed −4 Kcal (moderate search        strategy); the most stable dimer overall being less important;    -   in view of multiplex PCR, the following guidelines were taken        into account: a common primer would have to be designed in the        most consensus region (i.e. high PE in consensus search),        whereas individual primers (family or member) have to be        designed in the least consensus region (i.e. low PE value of        that primer for gene segments that should not be covered) to        avoid cross-annealing to other gene segments and thereby        multiple (unwanted) PCR products.

PCR Protocol

A standardised PCR protocol was developed based on pre-existingexperience from earlier European collaborative studies. After initialtesting and approval, the protocol was accepted as summarized in Table3.

Techniques for Analysis of PCR Products Obtained from Ig/TCR GeneRearrangements

The PCR products obtained from Ig and TCR gene rearrangements have to beanalysed for discrimination between monoclonal lymphoid cells withidentical junctional regions and polyclonal lymphoid cells with highlydiverse junctional regions.

Based on the combined experience of the participating laboratories, twotechniques were selected: heteroduplex (HD) analysis and Gene Scanning(GS) analysis. HD analysis uses double-stranded PCR products and takesadvantage of the length and composition of the junctional regions,whereas in GS single-stranded PCR products are separated in a highresolution gel or polymer according to their length only (FIG. 2).

Heteroduplex Analysis of PCR Products

PCR products obtained with unlabeled primers are denatured at hightemperature (˜95° C. for 5 min), followed by rapid random renaturationat low temperature (preferably at 4° C. for 1 hour). This enforcedduplex formation results in many different heteroduplexes with differentmigration speed in case of polyclonal lymphoproliferations, butresulting in homoduplexes with identical rapid migration in case ofmonoclonal lymphoproliferations. Electrophoresis of the homoduplexes ina 6% polyacrylamide gel results in a single band of predictable size,whereas the heteroduplexes form a smear at a higher position (FIG. 2).The heteroduplex technique is rapid, simple and cheap (see Table 4 fortechnical details) and has a detection limit of ˜5%.^(40, 41) Thedetection limit is influenced by the frequency of polyclonallymphocytes, because the formation of many heteroduplexes will alsoconsume a part of the monoclonal PCR products.”

Genescanning Analysis of PCR Products

The PCR primers for GeneScanning analysis need to be labeled with afluorochrome to allow detection of the PCR products with automatedsequencing equipment (FIG. 2).

The fluorochrome labeled single-strand (denatured) PCR products aresize-separated in a denaturing polyacrylamide sequencing gel orcapillary sequencing polymer and detected via automated scanning with alaser (see Table 5 for technical details). This results in a Gausiandistribution of multiple peaks, representing many different PCR productsin case of polyclonal lymphoproliferations, but gives a single peakconsisting of one type of PCR product in case of a fully monoclonallymphoproliferation (FIG. 2).

GeneScanning is rapid and relatively simple, but needs expensiveequipment. GeneScanning is generally more sensitive than heteroduplexanalysis and can reach sensitivities of 0.5 to 1% of clonal lymphoidcells.

Control Genes and Paraffin-Embedded Tissues

In several European countries, fresh tissue material is not easilyavailable for molecular diagnostics such as PCR-based clonality studies.Therefore one of the aims of the present study was to develop a strategyfor PCR-based clonality studies in paraffin-embedded tissues.

To control for the quality and amplifiability of DNA fromparaffin-embedded material, a special multiplex control gene PCR wasdeveloped, resulting in a ladder of five fragments (100 bp, 200 bp, 300bp, 400 bp, and 600 bp). From 45 of the above described 90 casessufficient paraffin-embedded tissue was available for DNA extraction.These DNA samples were tested in parallel to the freshly-obtained DNAsamples, using the Control Gene multiplex tube as well as theIg/TCR/BCL1/BCL2 multiplex tubes for clonality diagnostics (see Example10).

EXAMPLE 1 Complete IGH Gene Rearrangements: V_(H)J_(H) Background

The functional rearrangement of the IGH gene, first D_(H) to J_(H) andsubsequently V to is followed by antibody expression, the hallmark ofmature B-cells. The IGH gene is located on chromosome 14q32.3 in an areacovering approximately 1250 kilobases. 46 to 52 functional V_(H)segments (depending on the individual haplotype) have been identified,which can be grouped according to their homology in six or seven V_(H)subgroups. In addition approximately 30 non-functional V_(H) segmentshave been described. Furthermore, 27 D_(H) segments and functional sixJ_(H) segments have been consistently found (Table 2 and FIG. 3A).⁵⁶

The V_(H) segments contain three framework (FR) and two complementaritydetermining regions (CDR) (FIG. 3B). The FRs are characterized by theirsimilarity among the various V_(H) segments whereas the CDRs are highlydifferent even within the same V_(H) family. Furthermore, the CDRsrepresent the preferred target sequences for somatic hypermutations inthe course of the germinal center reaction, which increase thevariability within those regions. Although the FRs are usually lessaffected by somatic mutations, nucleotide substitutions may also occurwithin these regions, especially in B-cells under a heavy mutationalprocess.

The highly variable V-D-J_(H) regions can be amplified by PCR to detectclonal B-cell populations indicative of the presence of a malignantB-cell disorder. Clonal B-cells can be discriminated from polyclonalB-cells (i.e. normal or reactive lymphoid tissues) based on theidentical size and composition of the clonal PCR products as compared tothe many different polyclonal PCR products with a size range ofapproximately 60 bp, arranged in a Gaussian distribution. PCR-basedstrategies for detection of clonal B-cell populations in histologicalsections and cell suspensions have already been established in the earlynineties. However, the initial PCR protocols used single V_(H) consensusprimers which were able to bind to one of the three framework regions,mainly FR3. Such consensus primers were not suitable to amplify allV_(H) segments with the same efficiency leading to non-detectability ofa significant number of clonal rearrangements. In addition, somaticmutations introduced in the course of the germinal center reaction arenot restricted to the CDRs, but can also occur in FRs, therebypreventing primer annealing and consequently leading to absence ofclonal PCR products despite the presence of a neoplastic B-cellpopulation. This is especially true for follicular lymphomas, diffuselarge B-cell lymphomas, and multiple myelomas which usually contain highnumbers of somatic mutations.

To further increase the detection rate of the IGH PCR, several attemptshave been made to design family-specific primers to overcome thelimitations of consensus primers. However, these family-specific primersare largely based on the sequences of the previous consensus primers.Although these PCR strategies have helped to improve the detection rate,there is still a need of primer systems which are less sensitive tosomatic hypermutations, thus allowing amplification of (virtually) allpossible V-D-J_(H) rearrangements.

Primer Design

According to the guidelines of the invention, three sets of V_(H)primers were designed with the help of the OLIGO-6.2 programcorresponding to the three V_(H) frame work regions (FR1, FR2 and FR3)(FIG. 3B). Each set of primers consisted of six or sevenoligonucleotides capable to anneal to their corresponding V_(H) segments(V_(H) ₁ to V_(H) ₇ ) with no mismatches for most V_(H) segments and oneor at most two mismatches for some rare V_(H) segments. The design wassuch that mismatches would be located at the very 5′-end of the primer.These V_(H) primer sets we're used in conjunction with a single J_(H)consensus primer, designed to anneal to the most homologous 3′-end ofthe six J_(H) segments, approximately 35 bp downstream of the J_(H) RSS.This ensures that all J_(H) segments are detectable with the samebinding efficiency and that the primer binding will not easily beaffected by extensive nucleotide deletion in the course of therearrangement process. In addition, there was no cross-annealing betweenthe V_(H) primers and the J_(H) primer as evaluated by the OLIGO-6.2program.

The J_(H) primer was also designed to be used for amplification of otherPCR targets, such as incomplete D_(H)-J_(H) rearrangements as well ast(11;14) (BCL1-IGH) and t(14;18) (BCL2-IGH). This allows the detectionof different PCR products by GS analysis employing the same labeledJ_(H) primer.

Results of Initial Testing Phase

The initial testing of the newly designed V_(H)-J_(H) PCR was done byseparate application of each V_(H) primer together with the J_(H) primerin an individual PCR. For this purpose, DNA extracted from B-cell linesas well as well-defined clonal patient samples was used. Furthermore,clonal rearrangements were tested for sensitivity by serial dilution inDNA extracted from reactive tonsils. Clonal control samples were notavailable for each possible IGH rearrangement, but all major V_(H)segments and several rarely rearranged V_(H) segments have been includedin the initial testing phase.

All primer pairs worked with high efficiency and sensitivity. Theexpected clonal V_(H) rearrangements were detectable and the sensitivitywas at least 1% (10⁻²). There was no background within the expected sizerange and the amplification of tonsillar DNA gave the expected Gaussiandistribution curve. (FIGS. 3C, D, and E)

Based on these results we started the next phase of the initial primertesting by combining the V_(H) primers into three sets, each specificfor one of the three framework regions, which were used together withthe common J_(H) primer (FIG. 3B). The results were the same as thoseobtained with single primer pairs, but with a slightly lowersensitivity. In addition, no nonspecific products were amplified withinthe expected size range, with the exception of a 340 bp PCR productwhich appeared in the FR1 multiplex PCR. This PCR product was generatedirrespective of the source of the DNA (lymphoid and non-lymphoid) usedfor PCR, whereas no PCR product was obtained when no DNA template wasapplied. Furthermore, this amplicon was only detectable in heteroduplexanalyis, not in GeneScanning. This indicates that the fluorescentlabeled J_(H) primer was not involved in the generation of this PCRproduct. Sequence analysis of this PCR product disclosed a V_(H)4fragment amplified by the FR1 V_(H)4 primer in conjunction with the FR1V_(H)2 primer which apparently acted as a downstream primer by bindingto the intronic V_(H)4 sequence. This problem could be solved bydesigning a new FR1 V_(H)2 primer which was located 25 bp upstream tothe previous primer binding site.

Results of General Testing Phase

The approved IGH PCR was applied to the 90 Southern blot defined DNAsamples, which were derived from well-characterized cases. Six of the 11laboratories involved in the general testing phase performed GS analysisof the PCR products and five performed HD analysis. In addition severalpolyclonal as well as monoclonal samples (cell line DNA) were includedas controls. 45 of these cases displayed dominant PCR products after GSanalysis and 40 cases after HD detection, indicating the presence of amonoclonal B-cell population. The clonal rearrangements were detectablewith all three FR primer sets in 33 of the 45 clonal cases (GS) and inthe remaining 12 with one or two of the three FR primer sets. It wasconcluded that most negative results were caused by somatichypermutations in the primer binding site, preventing primer annealingand thus amplification.

The comparison of the V_(H)-J_(H) PCR results with the Southern blotresults revealed a high degree of concordance. 85% (46 out of 55) and76% (42 out of 55) of the samples with rearranged V_(H) genes bySouthern blot analysis showed a dominant amplification product by GSanalysis and HD analysis, respectively. Vice versa, all but two samplesharboring germline V_(H) genes by Southern blot displayed a polyclonalpattern by GS and HD analysis.

Conclusion

In conclusion, the three multiplex PCRs for detection of clonalV_(H)-J_(H) rearrangements provide a new and reliable assay to identifyclonal B-cell proliferations. The combined use of standardized primersin the three different FRs helps to decrease the rate of false-negativeresults due to somatic hypermutation in primer binding sites of theinvolved V_(H) gene segments.

EXAMPLE 2 Incomplete IGH Gene Rearrangements: D_(H)-J_(H) Background

The formation of complete V-D-J rearrangements in the IGH locus onchromosome 14q32.3 is a sequential process that occurs in two steps:V_(H) coupling is generally preceded by an initial rearrangement betweenD_(H) and J_(H) gene segments in early precursor-B cells (reviewedby⁵⁷). In addition to the many distinct V_(H) gene segments and the sixfunctional J_(H) gene segments (see Example 1), the human IGH locus alsocontains 27 D_(H) gene segments.⁵⁸ Based on sequence homology, the 27D_(H) segments can be grouped into seven families: D_(H)1 (formerlyknown as DM), D_(H)2 (DLR), D_(H)3 (DXP), D_(H)4 (DA), D_(H)5 (DK),D_(H)6 (DN), and D_(H)7 (DQ52); all families comprise at least fourmembers, except for the seventh which consists of the single D_(H)7-27segment just upstream of the J_(H) region (FIG. 3A).⁵⁸ ⁵⁹

Recombination between any of the D_(H) and J_(H) segments will result inthe formation of incomplete D_(H)-J_(H) joints, which can easily bedetected in bone marrow-derived CD10⁺/CD19 precursor B-cells^(60, 61)and hence also in a subset (20-25%) of precursor B-cell acutelymphoblastic leukemias, which show an immature genotype.⁶² Sequencingrevealed a predominance of D_(H)2 (D_(H)2-2), D_(H)3 (D_(H)3-9), andD_(H)7-27 gene segments in precursor B-ALL, comprising 36%, 33%, and 19%of all identified segments, respectively. ⁶²

However, also in mature B-cell malignancies incomplete D_(H)-J_(H)rearrangements have been reported.^(61, 63) Moreover, even in a subsetof IgH-negative multiple myelomas, which can be considered as the mostmature type of B-lineage malignancy, D_(H)-J_(H) joints were observed.⁶⁴These D_(H)-J_(H) rearrangements were derived from the non-coding secondallele and involved segments from D_(H)1 to D_(H)4 families.⁵⁴ Based onthe description of D_(H)-J_(H) joints in precursor-B-ALL and multiplemyelomas, it is assumed that incomplete D_(H)-J_(H) rearrangements arealso present in other types of B-cell leukemias and lymphomas. Inimmature T-cell malignancies D_(H)-J_(H) couplings have been identifiedas cross-lineage rearrangements;³⁴ interestingly, these almostexclusively occurred in the more immature non-TCRαβ⁺ T-ALL subset andmainly involved the more downstream D_(H)6-19 and D_(H)7-27 segments.The latter segment is frequently (up to 40%) used in fetal B cells butrarely in adult B cells.^(65, 66) Human adult precursor and mature Bcells mainly seem to use D_(H)2 and D_(H)3 family segments, as evidencedfrom sequences of complete V_(H)-D_(H)-J_(H) rearrangements.⁶⁶

Although the exact frequencies of incomplete D_(H)-J_(H) couplings indifferent types of mature B-cell malignancies are largely unknown, it isclear that they will at least be lower than those of V_(H)-J_(H)joinings. Nevertheless, D_(H)-J_(H) rearrangements might still representan important complementary target for PCR-based clonality assessment.This presumed contribution of D_(H)-J_(H) rearrangements as PCR targetis based on the assumption that incomplete rearrangements in the IGHlocus will not to contain somatic hypermutations, because transcriptionstarting from the promoters in the V gene segments does not occur, whichis regarded as an essential prerequisite for somatic hypermutation totake place.^(67, 68) Especially in those types of B-lineageproliferations in which somatic hypermutations are frequent, PCRanalysis of a possible D_(H)-J_(H) recombination product might thereforebe relevant, and sometimes even the only possibility to detect theB-cell clone.

Primer Design

Based on the high degree of homology within each D_(H) family, sevenfamily-specific D_(H) primers were designed (FIG. 4) in combination withthe consensus J_(H) primer that is also used for detection ofV_(H)-J_(H) rearrangements (see Example 1) and t(11;14) (BCL1-IGH) andt(14;18) (BCL2-IGH) (Examples 8 and 9). Primers were designed such thatcross-annealing to other D_(H) family segments would be minimal orpreferably absent, resulting in distinct positions for the variousfamily primers relative to the RSS elements (FIG. 4). The expected PCRproduct sizes of D_(H)-J_(H) joints range from 110-130 bp (forD_(H)7-J_(H) joinings) to 395-415 bp (for D_(H)3-J_(H) rearrangements).Of note, due to the position of the D_(H)7-27 segment close to thesegments in the J_(H) region, PCR products of 211 bp (and also419,1031,1404,1804, and 2420 bp in case of primer annealing todownstream J_(H) gene segments) will be amplified from non-rearrangedalleles and will be detected as a ladder of germline bands in virtuallyevery sample.

Results of Initial Testing Phase

For initial testing of the individual D_(H) primers, high tumor loadprecursor B-ALL or T-ALL samples with well-defined clonal D_(H)-J_(H)rearrangements were used. Under standard PCR conditions using 1.5 mMMgCl₂ and AmpliTaq Gold buffer, all seven primer combinations appearedto detect the clonal D_(H)-J_(H) targets with product lengths within theexpected size ranges. Cross-annealing of the D_(H) primers to rearrangedgene segments from other D_(H) families was only very weak or notobserved at all. Furthermore, also in healthy control tonsillar or MNCDNA PCR products of the correct size ranges were observed. Nonspecificannealing of the primers was not observed for virtually all primerssets, using non-template specific control DNA; only in case of theD_(H)2/J_(H) primer set a (sometimes faint) 340-350 bp product wasobserved in HeLa DNA. Further sequencing revealed that this nonspecificproduct was due to false priming of the D_(H)2 primer to a DNA sequenceupstream of the J_(H)4 segment. However, as the size of this nonspecificproduct was so different from the sizes of any of the true D_(H)-J_(H)PCR products, it was decided not to design a new D_(H)2 primer. In fact,the nonspecific 350 bp band can be employed as an internal marker forsuccessful DNA amplification and hence the quality of the template DNA,being hardly or only faintly visible when enough clonal or polyclonalD_(H)-J_(H) template is available (e.g. in tonsillar DNA or DNA fromparticular leukemic samples), but being especially strong in samplescontaining low numbers of lymphoid cells with D_(H)-J_(H)rearrangements.

Serial dilutions of DNA from the clonal reference samples into tonsillarDNA generally resulted in sensitivities of 5% or lower (0.5-1% in caseof the D_(H)6-J_(H) rearrangement) using HD analysis; sensitivities inGS analysis were generally 1-2 dilution steps better, i.e. 1% or lower.The clonal D_(H)7-J_(H) target could only be detected with a sensitivityof ˜10%, which is most probably caused by primer consumption in PCRamplicons involving the non-rearranged germline D_(H)7 and J_(H) genesegments.

Although the initial multiplex strategy, as suggested from the OLIGO6.2-assisted primer design, was to divide the various D_(H) primers overtwo tubes, it was decided after testing various multiplex approaches tocombine all primers into one multiplex tube (tube D of IGH clonalityassay), except for the D_(H)7 primer, which was included in a separatetube (tube E of IGH clonality assay). The reason to exclude the D_(H)7primer was the complicated germline pattern, due to easy amplificationof alleles with non-rearranged D_(H)7 segments. Using this two-tubemultiplex approach, all clonal reference samples were still detectable.Under multiplex conditions the detection limits for these various clonaltargets were logically less optimal as compared to the single assays,ranging from ˜5% (D_(H)3, D_(H)4, and D_(H)6) to ˜10% (D_(H)2, andD_(H)5). For the D_(H)1 clonal reference sample that was available, asensitivity of ˜20% was observed; at a later stage the D_(H)1-J_(H)rearrangement of cell line KCA was found to be detectable down to 10% inthe multiplex assay. As tube E only contains the D_(H)7 primer, the 10%sensitivity for this tube was the same as mentioned before. The samemultiplex analysis performed with 500 ng instead of 100 ng DNA of theserial dilutions, resulted in slightly better sensitivities. The use ofserial dilutions in MNC DNA instead of tonsillar DNA did not clearlyaffect detection limits of the assays for D_(H)-J_(H) recombinations.

Results of General Testing Phase

Following initial testing in the three laboratories involved in primerdesign, the developed IGH D_(H)-J_(H) multiplex PCR assay was furtherevaluated using the 90 Southern blot-defined samples. Every sample wasanalyzed in parallel in four laboratories by HD analysis and in fivelaboratories by GS analysis; in another two laboratories all sampleswere analyzed by both techniques. All together a total of six HD andseven GS analysis results were obtained per sample per tube. Despiteconcordant results (>80% of laboratories with identical results) in thevast majority of samples, nine showed inter-laboratory discordancies intube D. Further analysis revealed that these could be explained byeither the presence of a small clone with weak clonal products, or tolarge size products (˜390 and larger). In a few cases the products wereso large, that only after sequencing it became clear that they concernedtrue but extended D_(H)-J_(H) rearrangements, either from upstream D_(H)(e.g. D_(H)6-25-D_(H)1-26-J_(H) in NL-12) or from downstream J_(H) genesegments (e.g. D_(H)6-25-JH4-J_(H)5 in PT-14). In all three cases(NL-17, mycosis fungoides; FR-1, B-CLL; FR-5, FCL) in which clonalproducts were found using tube E, the results were completely concordantbetween laboratories.

When evaluating results from HD and GS analysis, it appeared that thesewere comparable, although in general the number of laboratories showingidentical results was slightly higher upon HD as compared to GS analysis(FIGS. 4B and C).

Direct comparison of D_(H)-J_(H) multiplex PCR results with SB data isvirtually impossible, as hybridization with a single probe (IGHJ6) inthe J_(H) region does not allow discrimination between V_(H)-J_(H) andD_(H)-J_(H) rearrangements. In three samples it was clear that detectionof clonal products of the combined VH-J_(H) and D_(H)-J_(H) assays didnot fit with configuration of the IGH locus in SB analysis. Remarkably,no clonal D_(H)-J_(H) PCR products were observed in the pre-follicularB-cell malignancies. In contrast, 11/16 B-CLL samples and 12/25(post-)follicular B-cell malignancy samples did contain clonallyrearranged D_(H)-J_(H) PCR products. In three of the eighteen T-cellmalignancy cases clonal D_(H)-J_(H) rearrangements were seen; theseconcerned T-LBL (ES-9) and mycosis fungoides (NL-17) cases withSB-detected IGH rearrangements, and a case of T-NHL/EATL (PT-4) withoutSB-detected IGH rearrangements, probably because of the low tumor loadof <15%. All 15 reactive cases only showed polyclonal D_(H)-J_(H) PCRproducts, in accordance with SB results. In category D with difficultdiagnoses, three samples (PT-12, GBS-10, and GBN-8) showed clonal IGHD_(H)-J_(H) PCR products, which was in line with SB data as well as IGKPCR data in two of three cases; in another two samples (PT-6 and GBS-9),both T-cell rich B-NHL cases, clonal D_(H)-J_(H) products were found inaddition to clonal IGK and/or IGL products, but without evidence forclonality from SB analysis, which might best be explained by the smallsize of the B-cell clone in these samples.

In order to determine the additional value of D_(H)-J_(H) PCR analysis,the results were compared to those of V_(H)-J_(H) PCR analysis. In five(NL-4, PT-14, GBN-2, FR-7, NL-12) B-cell malignancies clonal D_(H)-J_(H)PCR products were found, whereas only polyclonal V_(H)-J_(H) PCRproducts were observed.

Conclusion

In conclusion, based on the initial and general testing phases,D_(H)-J_(H) PCR analysis appears to be of added value for clonalityassessment. Although HD analysis results might be interpreted slightlymore easily, there is no clear preference for either HD or GS analysisas they are both suitable for analyzing amplified PCR products. Apotential difficulty in D_(H)-J_(H) PCR analysis is the relatively largesize range of expected PCR products, due to scattered primer positionsand to extended amplifications from upstream D_(H) or downstream J_(H)gene segments, implying that long runs are recommended for GS analysis.Finally, the remarkable position of the D_(H)7-27 gene segment in theIGH locus causes a ladder of germline amplification products in tube E,with clonal products being easily recognizable as much smallerbands/peaks.

EXAMPLE 3 IGK Gene Rearrangements: Vκ-Jκ, Vκ-Kde/intronRSS-KdeBackground

The human IGK light chain locus (on chromosome 2p11.2) contains manydistinct Vκ gene segments, grouped into seven Vκ gene families, as wellas five Jκ gene segments upstream of the Cκ region. Originally, the Vκgene segments were designated according to the nomenclature as describedby Zachau et al.⁶⁹ An alternative nomenclature groups the Vκ genesegments in seven families and is used in the ImMunoGeneTics database.⁴⁶Here we follow the latter nomenclature. The Vκ1, Vκ2, and Vκ3 familiesare multi-member families including both functional and pseudo genesegments, whereas the other families only contain a single (Vκ4, 78 5,Vκ7) or a few segments (Vκ6).⁷⁰ Remarkably, all Vκ gene segments aredispersed over two large duplicated clusters, one immediately upstreamand in the same orientation as the Jκ segments, and the other moredistal and in an inverted orientation (FIG. 5A).⁷¹ The latter impliesthat so-called inversion rearrangements are required to form Vκ-Jκjoints involving Vκ genes of the distal cluster. In addition to the Vκand Jκ segments, there are other elements in the IGK locus that can beinvolved in recombination. The kappa deleting element (Kde),approximately 24 kb downstream of the Jκ-Cκ region, can rearrange to Vκgene segments (Vκ-Kde), but also to an isolated RSS in the Jκ-Cκ intron(intronRSS-Kde).^(24, 72) Both types of rearrangements lead tofunctional inactivation of the IGK allele, through deletion of eitherthe Cκ exon (intronRSS-Kde rearrangement) or the entire Jκ-Cκ area(Vκ-Kde rearrangement).

As human IGK recombination starts in precursor B-cells in the bonemarrow, IGK rearrangements can also be detected in precursor B-cellacute leukemias (30-45% of alleles, depending on age). Although Vκ-Jκjoinings are present, these IGK rearrangements mainly concernrecombinations involving Kde (25-35% of alleles). In childhood precursorB-ALL Vκ-Kde recombination predominates over intron-Kde, whereas inadult ALL the deletions exclusively concern Vκ-Kdecouplings.^(24, 73, 74) In chronic B-cell leukemias IGK rearrangementsare even more frequent, being detectable on 75% (Ipκ⁺ cases) or even 95%(Igλ⁺ cases) of all IGK alleles. By definition, functional Vκ-Jκrearrangements are found on at least one allele in Igκ⁺ B-cellleukemias; the non-coding second allele is either in germlineconfiguration, or inactivated through Vκ-Kde (8% of alleles) orintronRSS-Kde (8% of alleles) recombination. Kde rearrangements arefrequent in Igλ⁺ B-cell leukemias (˜85% of alleles), with a slightpredominance of intronRSS-Kde recombinations over Vκ-Kde rearrangements.This implies that virtually all Igκ⁺ leukemias contain a Kderearrangement, while potentially functional Vκ-Jκ couplings arerelatively rare.^(24, 75) Several studies have shown that Vκ genesegment usage is almost identical between various normal and malignantB-cell populations and largely reflects the number of available genesegments within each family. Both in Vκ-Jκ as well as in Vκ-Kderearrangements, Vκ gene segments from the first four families (Vκ1 toVκ4) predominate. Vκ2 gene usage appeared to be higher in precursorB-ALL than in more mature B-cell lyinphoproliferations or normal Bcells. Remarkably, the distal inverted Vκ cluster was rarely used inVκ-Jκ rearrangements, whereas Vκ pseudogene segments were neverinvolved, also not in Igλ⁺ cases.³⁶ Little is known about Jκ genesegment usage, but sparse data show that Jκ1, Jκ2, and Jκ4 are the mostfrequently used Jκ gene segments.⁷⁵

Vκ-Jκ rearrangements can be important complementary PCR target for thosetypes of B-cell proliferations in which somatic hypermutations mayhamper amplification of the V_(H)-J_(H) target, but recombinationsinvolving Kde are probably even more valuable. Deletion of interveningsequences in the Jκ-Cκ intron results in the removal of the IGKenhancer, which is thought to be essential for the somatic hypermutationprocess to occur. Rearrangements involving Kde are therefore assumed tobe free of somatic hypermutations, and hence should be amplified rathereasily.

Primer Design

Using OLIGO 6.2 software, six family-specific Vκ primers were designedto recognize the various Vκ gene segments of the seven Vκ families; theVκ6 family gene segments were covered by the Vκ1 family primer (FIG.5B). In case of the relatively large Vκ1, Vκ2, and Vκ3 families only thefunctional Vκ gene segments were taken into consideration, as the lesshomologous pseudo gene segments complicated optimal primer design toomuch. The family-specific Vκ primers were designed to be used incombination with either a set of two Jκ primers (Jκ1-4, covering thefirst four Jκ segments and Jκ5 covering the fifth) or a Kde primer (FIG.5B). For analysis of Kde rearrangements an additional forward primerrecognizing a sequence upstream of the intronRSS was made. In order toshow minimal cross-annealing to other Vκ family segments and still beuseful in multiplex reactions, the various primers could not be designedat similar positions relative to RSS elements (FIG. 5B). The expectedPCR product sizes of Vκ-Jκ joints range from ˜115-135 bp (for Vκ7-Jκjoints) to ˜280-300 bp (Vκ2-Jκ rearrangements). For the Kderearrangements, product size ranges are from ˜495-215 bp (Vκ7-Kde) to˜360-380 bp (Vκ2-Kde), whereas the intronRSS-Kde products are ˜275-295bp.

Results of Initial Testing Phase

For initial testing of the individual primers, several cell lines andpatient samples with precisely defined clonal Vκ-Jκ, orVκ-Kde/intronRSS-Kde rearrangements were used. The patient samples withVκ-Jκ joints mostly concerned chronic B-cell leukemias, which wereadditionally selected on basis of a high tumor load for easy andsensitive detection of the involved rearrangement. Unfortunately, clonalreference samples were not available for all Vκ-Jκ targets; especiallythe more rare types of rearrangements involving VκK5, Vκ7 and/or Jκ5were not represented in the series of reference samples. For thesetargets and also for the targets for which clonal reference samples wereavailable, healthy control tonsillar or MNC DNA samples were employed,in which PCR products of the correct expected sizes were indeedobserved. The only exception was the Vκ7/Jκ5 primer combination; mostprobably Vκ7-Jκ5 joinings are so rare in normal B cells, that these PCRproducts were hardly or not detectable in tonsils. Rearranged productswithin the expected size ranges could be detected in all clonalreference samples, under standard PCR conditions using 1.5 mM MgCl₂ andeither ABI Gold Buffer or ABI Buffer II. However, in a few cases weakamplification of particular Vκ-Jκ rearrangements was observed with otherVκ family/Jκ primer sets, due to slight cross-annealing of the Vκ3primer to a few Vκ1 gene segments. Furthermore, in a few of the clonalreference samples clear additional clonal PCR products were seen withother Vκ/Jκ or even Vκ/Kde and intronRSS/Kde primer sets; in mostsamples this could be explained by the complete configuration of the twoIGK alleles. This occurrence of multiple clonal PCR products illustratesthe complexity of IGK rearrangement patterns in a given cell sample,mainly caused by the potential occurrence of two clonal rearrangementson one allele (Vκ-Jκ and intron RSS-Kde). This complexity does nothamper but support the discrimination between polyclonality andmonoclonality.

No nonspecific annealing of the primers was observed for any of theVκ-Jκ and Vκ-Kde/intron RSS-Kde primer sets, when using HeLa DNA as anon-template specific control. Serial dilutions of DNA from the clonalreference samples into tonsillar DNA generally resulted in sensitivitiesof 5-10% for Vκ-Jκ rearrangements and 1-10% for Vκ-Kde rearrangements,using HD analysis. In general, the sensitivities in GS analysis wereapproximately one dilution step better. The only slightly problematictarget was the intronRSS-Kde target that could only be detected down tothe 10% serial dilution in the employed patient sample. This is probablycaused by the fact that intronRSS-Kde rearrangements are abundant in DNAfrom both Igκ⁺ and Igλ⁺ tonsillar B cells, which were used in thedilution experiments.

The multiplex strategy that was chosen after testing several approachesconsisted of two different multiplex PCR reaction tubes. In the Vκ-Jκtube (tube A) all Vκ primers were combined with both Jκ primers, whereastube B contained all Vκ primers plus the intronRSS primer in combinationwith the Kde reverse primer (FIG. 5B). All beforementioned clonalreference samples were detectable using this two-tube multiplexapproach. Of note is the observation that in non-clonal tonsil samples apredominant, seemingly clonal band of ˜150 bp was detected using theVκ-Jκ multiplex tube A analysis. The presence of this product, which isseen in HD analysis but especially in GS analysis, can be explained bythe limited heterogeneity of Vκ-Jκ junctional regions leading to a highfrequency of products of an average size of ˜150 bp. Furthermore, insome samples a sometimes weak 404 bp nonspecific band was observed intube B. Although sensitivities were on average slightly better in othermultiplex approaches in which the Vκ primers were further subdividedover multiple tubes, the feasibility of having only two tubes to analyzeall relevant IGK rearrangements, finally was the most important argumentfor choosing the two-tube multiplex strategy as given in FIG. 5B.Detection limits for the various clonal targets in the two-tubemultiplex approach were ˜10% for most of the clonal Vκ-Jκ rearrangements(Vκ1-Jκ4, Vκ2-Jκ4, Vκ3-Jκ4) derived from informative samples with a hightumor load; several of the Vκc-Kde targets were detectable with a stillreasonable sensitivity of ˜10%, but a few other samples containingVκ2-Kde, Vκ5-Kde, and also intronRSS-Kde targets showed detection limitsabove 10%. Even the use of 500 ng serially diluted DNA instead of 100 nghardly resulted in better sensitivities, whereas serial dilutions in MNCDNA did not affect the detection limits either. Nevertheless, detectionlimits of serial dilutions of reference DNA in water were all in theorder of 0.5-1%, which shows that the chosen multiplex IGKPCR assay assuch is good. It is important to note that potential clonal cellpopulations in lymph nodes or peripheral blood in practice will have tobe detected within a background of polyclonal cells, which can hampersensitive clonality detection, especially in samples with a relativelyhigh background of polyclonal B-cells.

Results of General Testing Phase

Following initial testing in the four laboratories involved in primerdesign, the developed IGK multiplex PCR assay was further evaluatedusing 90 Southern blot-defined samples. Every sample was analyzed inparallel via HD (five laboratories) and GS (two laboratories) analysis;in another four laboratories all samples were analyzed by bothtechniques. Taken together, eight HD and five GS analysis results wereavailable per sample per tube. In the vast majority of samples >80% oflaboratories produced identical results, i.e. either clonal bands/peaksor polyclonal smears/curves in one or both tubes. However, in nine(˜10%) samples discordancies were found between laboratories, whichremained after repetitive analysis of these samples. More detailedanalysis revealed that in at least six cases the approximately 150 and200 bp sizes of the clonal products in tube A could not easily bediscriminated from polyclonal products of roughly the same size. This isan inherent difficulty in especially Vκ-Jκ analysis, which is caused bythe relatively limited junctional heterogeneity of these rearrangements.In two samples the results from tube B were however so clear in alllaboratories with both techniques that in fact no discrepancy prevailed.In one sample (ES-8) a large product of around 500 bp appeared to be thereason for discrepant inter-laboratory results; further sequencingrevealed that amplification starting from the downstream Jκ segmentcaused production of an extended Vκ1-Jκ3-Jκ4 PCR product.

When evaluating results from HD and GS analysis, it appeared that thesewere rather comparable, although in general the number of laboratoriesshowing identical results was slightly higher upon HD as compared to GSanalysis (FIGS. 5C and D). Remarkably, in one sample (GBS-4) HD analysisrevealed a clear product in both tubes, whereas GS analysis only showedpolyclonality. Cloning of the HD product showed a peculiar Vκ3-Vκ5 PCRproduct, which was not observed in any other sample; the Vκ-Vκconfiguration of this product explained why it was not detected withlabeled Jκ primers in GS analysis.

Comparison of PCR results with SB data revealed no SB-PCR discrepanciesin the pre-follicular B-cell malignancies and B-CLL samples; in linewith the presence of rearranged IGK bands in SB analysis, all samplescontained clonal IGKPCR products. In contrast, in the 25(post-)follicular B-cell malignancy samples clonal IGK PCR products weremissed in four DLCL cases (ES-5, PT-13, PT-14, FR-7) and one PC leukemia(NL-19) with both techniques and in another DLCL case (GBS-4, see above)with GS analysis only. In all cases this was most probably caused bysomatic hypermutation. Interestingly, in one FCL case (NL-4), a clonalPCR product was found, whereas SB analysis revealed a germline band incase of the IGK genes and weak clonal bands upon IGH analysis. In all 18T-cell malignancy cases and all 15 reactive cases (category C)polyclonal IGKPCR products were found in accordance with SB results,except for one peripheral T-NHL case (FR-10). Next to the clonal TCR andIGK products this sample also showed clonal IGH and IGL PCR products,but no clonal Ig rearrangements in SB analysis, probably reflecting thepresence of a small additional B-cell clone in this sample. Finally, inthe category with difficult diagnoses (D), two samples (GBS-10 andGBN-8) showed clonal IGKPCR products, in line with SB data; however, inanother two samples (PT-6 and GBS-9), both T-cell rich B-NHL cases,clonal IGKPCR products were found as well as clonal IGH and/or IGLproducts, but without evidence for clonality from SB analysis. Also thisdiscrepancy can probably be explained by the small size of the B-cellclone in these two patient samples.

To determine the additional value of analyzing the IGK locus, wecompared the results of IGK PCR analysis to those of IGH PCR analysis.In five (ES-2, NL-4, PT-8, GBN-2, ES-8) of the nine samples in which noclonal V_(H)-J_(H) PCR products were found, clonal products were readilyobserved in IGK analysis. When taking into account both V_(H)-J_(H) andD_(H)-J_(H) analysis, IGKPCR analysis was still complementary to IGHPCRanalysis in three of these cases in detecting clonal Ig PCR products.

Conclusion

In conclusion, based on the initial and general testing phases as wellas preliminary evidence from use of these multiplex assays inpathologically well-defined series of lymphoproliferations, PCR analysisof the IGK locus has clear (additional) value for clonality detection.Nevertheless, care should be taken with interpretation of seeminglyclonal bands in especially tube A, due to the inherent restricted IGKjunctional heterogeneity. As this problem is especially apparent in GSanalysis, HD analysis is slightly preferred over GS analysis, althoughit should be marked that in some cases GS analysis may facilitate properinterpretation of results. Another potential pitfall is the relativelylarge size range of expected rearranged IGK products, due to scatteredprimer positions, and to extended amplifications from downstream Jκ genesegments. This implies that long runs are recommended for GS analysis.Finally, the inherent complexity of multiple rearrangements in the IGKlocus (Vκ-Jκ and Kde rearrangements on the same allele), together with alow level of cross-annealing of Vκ primers, may occasionally result inpatterns with multiple bands or peaks, resembling oligoclonality.However, with these considerations in mind, the two-tube IGK multiplexPCR system can be valuable in PCR-based clonality diagnostics.

EXAMPLE 4 IGL Gene Rearrangements Background

IGL gene rearrangements are present in 5 to 10% of Igκ⁺ B-cellmalignancies and in all Igλ⁺ B-cell malignancies.⁷⁵ Therefore Vλ-Jλrearrangements potentially represent an attractive extra PCR target forclonality studies to compensate for false-negative IGH V_(H)-J_(H) PCRresults, mainly caused by somatic mutations. The IGL locus spans 1 Mb onchromosome 22q11.2.^(77, 79) There are 73-74 Vλ genes over 900 kb, amongwhich 30-33 are functional (FIG. 6A). Upon sequence homology, the Vλgenes can be grouped in 11 families and three clans. Members of the samefamily tend to be clustered on the chromosome. The Jλ and Cλ genes areorganized in tandem with a Jλ segment preceding a Cλ gene. Typicallythere are 7 J-Cλ gene segments, of which J-Cλ1, J-Cλ2, J-Cλ3, and J-Cλ7are functional and encode the four Igλ isotypes (FIG. 6A).^(80, 81)There is however a polymorphic variation in the number of J-Cλ genesegments, since some individual may carry up to 11 of them, due to anamplification of the Cλ2-Cλ3 region.^(82, 83)

Several studies have shown that the IGL gene repertoire of both normaland malignant B cells is biased.^(48, 49, 84, 85) Thus over 90% of Vλgenes used by normal B cells belong to the Vλ1, Vλ2 and Vλ3 families,which comprise 60% of the functional genes. Moreover, three genes (2-14,1-40, 2-8) account for about half of the expressed repertoire. Whilenormal B cells use J-Cλ1, J-Cλ2 and J-Cλ3 gene segments in roughlyequivalent proportions, neoplastic B cells tend to use predominantlyJ-Cλ2 and J-Cλ3 gene segments.⁴⁹ In both normal and malignant B cellsthe J-Cλ7 is used very rarely (1%). This latter finding was howeverchallenged by a single-cell study of normal cells which found that morethan half of the rearrangements employed the J-Cλ7 gene segments.⁸⁶ Incontrast to the mouse, there is some junctional diversity due toexonuclease activity and N nucleotide addition in human IGL generearrangements.^(82, 85-87) It is however much less extensive than thatof the IGH locus, and a number of rearrangements result from thedirectly coupling of germline Vλ and Jλ a gene segments. Nevertheless,the IGL locus might represent an alternative complementary locus to IGHfor B-cell clonality studies.

Primer Design

Considering the biased Vλ repertoire, we chose to amplify onlyrearrangements which used the Vλ1, Vλ2 and Vλ3 gene segments. A singleconsensus primer recognizing both Vλ1 and Vλ2 gene segments, as well asa Vλ3 primer, were designed in regions of high homology between membersof the same family (FIG. 6B). Initial experiments showed that theyworked as well in multiplex as separately. In fact, cross annealing ofVλ3 primer hybridizing to some Vλ1 or Vλ2 genes (or vice versa) could beobserved when Vλ primers were used separately; it was not seen howeverin multiplex PCR.

A single consensus primer was designed for the Jλ1, Jλ2 and Jλ3 genesegments and has one mismatch in its central portion compared to each ofthe germline sequences. In preliminary experiments it was found to giverather better results than a combination of perfectly matched Jλ1 andJλ2-Jλ3 primers. Since a single study reported the frequent usage of theJλ7 gene in normal B cells,⁸⁶ we also designed a Jλ7 specific primer.When tested on various polyclonal B cell samples, we could hardly detectany signal in HD analysis, in contrast to amplifications performed onthe same samples using the Jλ1, Jλ2-Jλ3 or the Jλ consensus primers.Similarly, we could not detect any rearrangement with this primer whenanalyzing a collection of monoclonal B-cell tumors. Based on theseresults as well as the other reports in the literature⁴⁹, we concludedthat the non-confirmed high frequency of Jλ7 rearrangements (in a singlestudy)⁸⁶ had been caused by a technical pitfall and consequently, wedecided not to include the Jλ7 primer. The PCR assay for the detectionof IGL gene rearrangements in clonality study therefore consists of asingle tube containing three primers (FIG. 6B). This single tube wasexpected to detect the vast majority of the rearrangements.

Results of Initial Testing Phase

Initial testing on a set of monoclonal and polyclonal samples showedthey could very well be differentiated upon HD analysis of PCR productson 10% polyacrylamide gel electrophoresis (FIG. 6C). Clonal IGLrearrangements were seen in the homoduplex region, with one or sometimestwo weaker bands in the heteroduplex region, while polyclonalrearrangements appeared as a smear in the heteroduplex region (FIG. 6C).Nonspecific bands were not observed. It should be noted that because ofthe limited size of the junctional region, it is extremely difficult todistinguish polyclonal from monoclonal rearrangements by running asimple polyacrylamide gel without performing a heteroduplex formation.Along this line, analysis of PCR products by GS proved to be lessstraightforward (FIG. 6C). While monoclonal rearrangements were clearlyidentified, the polyclonal rearrangement pattern had an oligoclonalaspect due to the limited junctional diversity. The interpretation wasmore difficult, particularly to distinguish polyclonal cases from thosewith a minor clonal B-cell population in a background of polyclonalB-cells. We therefore recommend HD analysis as the method of choice toanalyze IGL gene rearrangements.

The sensitivity of the assay, performed on several cases, proved to beabout 5% (2.5%-10%) when dilution of tumor DNA was done in PB-MNC andabout 10% (5%-20%) when diluted in lymph node DNA.

Results of General Testing Phase

The single-tube IGL PCR assay was evaluated on the series of 90 Southernblot defined lymphoid proliferations. This testing was done by ninelaboratories, four with HD analysis only, one with GS analysis only, andfour using both techniques. Clonal IGL gene rearrangements were detectedin 19 cases. In 15 of them more than 70% concordance was obtained withinthe nine laboratories. In four cases less than 70% concordancy wasobtained, which could be explained by minor clonal IGL generearrangements in three of them (ES-12, GB-10, and FR-10). Thisdiscordancy in the fourth case (PT-11) remains unexplained, particularlybecause no IGL gene rearrangements were detected by Southern blotting.As concluded from the initial testing, interpretation of GS analysis wasmore difficult than HD analysis, especially in the case of minor clonalpopulations. Of these 19 clonal IGL gene cases, 17 were B-cellproliferations (16 mature and one precursor B-cell). One case (ES12)corresponded to Hodgkin's disease and another (FR-10) to a T-NHL. Bothhad only a minor clonal IGL gene rearrangement, and FR-10 also displayeda clonal IGK gene rearrangement.

Comparison with Southern blot data showed some discrepancies. Six caseswith clonal IGL gene rearrangements by PCR appeared as polyclonal bySouthern blot analysis. Three of them (PT-6, ES-12, FR-10) concernedminor clonal populations which may have been below the sensitivity levelof the Southern blot technique. In the three other cases (NL-19, ES-1,PT-11) a clonally rearranged band may have been missed by the fairlycomplex rearrangement pattern of the IGL locus on Southernblot.^(26, 49) Conversely the PCR assay failed to detect clonalrearrangements which were seen by Southern blot analysis in two cases(GBS-6, FR-5). However these were follicular lymphomas in which a highdegree of somatic hypermutations may have prevented annealing of the IGLgene primers.

Conclusion

In conclusion, a single-tube PCR assay for the detection of IGL generearrangements containing only three primers (FIG. 6B) allows to detectthe vast majority of IGL gene rearrangements (Vλ1, Vλ2, and V3 generearrangements). Heteroduplex analysis is the preferred analytic method,though GeneScan analysis can be used, but maximal caution is recommendedto avoid overinterpretation of clonality due to the limited junctionaldiversity.

EXAMPLE 5 TCRB Gene Rearrangements: Vβ-Jβ, Dβ-Jβ Background

Molecular analysis of the TCRB genes is an important tool for assessmentof clonality in suspect T-cell proliferations. TCRB gene rearrangementsoccur not only in almost all mature T-cell malignancies but also inabout 80% of the CD3 negative T-cell acute lymphoblastic leukemias(T-ALL) and 95% of the CD3 positive T-ALL.²⁸ TCRB rearrangements are notrestricted to T-lineage malignancies as about one third ofprecursor-B-ALL harbor rearranged TCRB genes.³⁰ Their frequency is muchlower (0 to 7%) in mature B cell proliferations.²¹

The human TCRB locus is located on the long arm of chromosome 7, at band7q34 and spans a region of 685 kb. In contrast to the TCRG and TCRD locithe V region gene cluster of the TCRB locus is far more complex (FIG.7A).¹ It contains about 65 Vβ gene elements for which two differentnomenclatures are used: the one summarized by Arden et al.⁵⁰ follows thegene designation of Wei et al.⁸⁸ and groups the Vβ genes into 34families. The alternative nomenclature proposed by Rowen et al.⁵¹subdivides 30 Vβ gene subgroups and was later adopted by IMGT, theinternational ImMunoGeneTics database http://imgt.cines.fr (initiatorand coordinator: Marie-Paule Lefranc, Montpellier, France). [Lefranc,2003 #212;Lefranc, 2003 #219] The largest families, Vβ5, Vβ6, Vβ8 andVβ13 (Arden nomenclature) reach a size of seven, nine, five and eightmembers, respectively. Twelve Vβ families contain only a single member.In general, the families are clearly demarcated from each other.⁵⁰ Inthis report we follow the Arden nomenclature.⁵⁰

39-47 of the Vβ gene elements are qualified as functional and belong to23 families. 7-9 of the nonfunctional Vβ elements have an open readingframe but contain alterations in the splice sites, recombination signalsand/or regulatory elements. 10-16 are classified as pseudogenes. Inaddition, a cluster of six non-functional orphan Vβ genes have beenreported that are localized at the short arm of chromosome 9(9p21).^(89, 90) They are not detected in transcripts.^(50, 51)

All but one Vβ genes are located upstream of two Dβ-Jβ-Cβ clusters. FIG.7A illustrates that both Cβ gene segments (Cβ1 and Cβ2) are preceded bya Dβ gene (Dβ1 and Dβ2) and a Jβ cluster which comprises six (Jβ1.1 toJβ1.6) and seven (Jβ2.1 to Jβ2.7) functional Jβ segments. Jβ region lociare classified into two families according to their genomiclocalization, not to sequence similarity.^(51, 88, 91)

Due to the large germline encoded repertoire, the combinatorialdiversity of TCRB gene rearrangements is extensive compared to the TCRGand TCRD rearrangements. The primary repertoire of the TCRβ molecules isfurther extended by an addition of an average of 3.6 (V-D junction) and4.6 (D-J junction) nucleotides and deletion of an average of 3.6 (V),3.8 (5′ of D), 3.7 (3′ of D) and 4.1 (J) nucleotides.⁵¹ The completehypervariable region resulting from the junction of the V, D and Jsegments comprises characteristically nine or ten codons. Size variationis limited, as 7 to 12 residues account for more than 80% of allfunctional rearrangements in contrast to the broad length repertoire ofthe IGH CDR3 region.⁹²

During early T-cell development the rearrangement of the TCRB geneconsists of two consecutive steps: Dβ to Jβ rearrangement and Vβ to D-Jβrearrangement with an interval of one to two days between these twoprocesses.⁹³ The Dβ1 gene segment may join either Jβ1 or Jβ2 genesegments but the Dβ2 gene segment generally joins only Jβ2 gene segmentsbecause of its position in the TCRB gene locus.^(28, 51) Due to thepresence of two consecutive TCRB D-J clusters, it is also possible thattwo rearrangements are detectable on one allele: an incomplete TCRBDβ2-Jβ2 rearrangement in addition to a complete or incompleterearrangement in the TCRB Dβ1-Jβ1 region.¹

In TCRB gene rearrangements, a non-random distribution of gene segmentusage is seen. In healthy individuals, some Vβ families predominate inthe peripheral blood T-cell repertoire (e.g Vβ1-Vβ5), while others areonly rarely used (e.g. Vβ11, Vβ16, Vβ18, Vβ23). Mean values of the Vβrepertoire seem to be stable during aging, although the standarddeviation increase in the elderly.^(13, 94) Also in the human thymussome Vβ gene segments dominate: the most prevalent seven Vβ genes(Vβ3-1, Vβ4-1, Vβ5-1, Vβ6-7, Vβ7-2, Vβ8-2, Vβ13-2) cover nearly half ofthe entire functional TCRB repertoire.⁹⁵ The representation of Jsegments is also far from even. The Jβ2 family is used more frequentlythan the Jβ1 family (72% vs. 28% of TCRB rearrangements).⁹⁶ Inparticular, the proportion of Jβ2.1 is higher than expected (24%)followed by Jβ2.2 (11%) and Jβ2.3 and Jβ2.5 (10% each).⁹⁵

TCRB gene rearrangement patterns differ between categories of T cellmalignancies. Complete TCRB Vβ-Jβ1 rearrangements and incompletelyrearranged alleles in the TCRB Dβ-Jβ2 cluster are seen more frequentlyin TCRαβ⁺ T-ALL as compared to CD3⁻ T-ALL and TCRγδ⁺ T-ALL.²⁸ In thetotal group of T-ALL the TCRB Dβ-Jβ1 region is relatively frequentlyinvolved in rearrangements in contrast to cross-lineage TCRB generearrangements in precursor-B-ALL which exclusively involve the TCRBDβ-Jβ2 region.^(30, 73)

The development of monoclonal antibodies against most Vβ domains hashelped to identify Vβ family expansions.¹³ However, TCR generearrangement analysis is essential for clonality assessment in T celllymphoproliferative disorders. As the restricted germline encodedrepertoire of the TCRG and TCRD loci facilitates DNA based PCRapproaches, various PCR methods have been established for the detectionof TCRG and TCRD gene rearrangements.⁹⁷⁻⁹⁹ Nevertheless, the limitedjunctional diversity of TCRG rearrangements leads to a high backgroundamplification of similar rearrangements in normal T cells (Example 6).The TCRD gene on the other hand is deleted in most mature T cellmalignancies.²¹ Therefore DNA based TCRB PCR techniques are needed forclonality assessment. In addition, TCRB rearrangements are of greatinterest for follow-up studies of lymphoproliferative disorders, becausethe extensive combinatorial repertoire of TCRB rearrangements and thelarge hypervariable region enables a highly specific detection ofclinically occult residual tumor cells. However, the extensive germlineencoded repertoire renders PCR assays more difficult. Some published PCRapproaches use the time consuming procedure of multiple tube approacheswith a panel of family- or subfamily-specific primers.^(96, 100) Usageof highly degenerated consensus primers limits the number of detectablerearrangements that are theoretically covered by the primers becausethere is no single common sequence of sufficient identity to allow areliable amplification of all possible rearrangements.^(42, 101, 102)Some published assays use a nested PCR requiring an additional PCRreaction.^(42, 102) Other assays focus on analysis of the TCRBVβ-Dβ-Jβ-Cβ transcripts to limit the number of primersneeded.^(16, 100, 103) However, a major drawback of these mRNA basedapproaches is the need for fresh or frozen material and a reiersetranscription step before the PCR amplification.

We tried to overcome these limitations by creating a completely new andconvenient DNA based TCRB PCR. We designed multiple Vβ and Jβ primers,covering all functional Vβ and Jβ gene segments and being suitable forcombination in multiplex PCR reactions. In addition the assay isapplicable for HD and GS analysis and also detects the incomplete TCRBDβ-Jβ rearrangements with the same set of Jβ primers. In order to avoidproblems due to cross priming we decided to design all Vβ and Jβ primersat the same conserved region of each gene segment.

Primer Design

Initially a total of 23 Vβ, 2 Dβ(Dβ1 and Dβ2) and 13 Jβ (Jβ1.1 to 1.6and Jβ2.1 to 2.7) primers were designed with all the Vβ and Jβ primerspositioned in the same conserved region of each Vβ and Jβ gene segmentso that the effects of cross-annealing in a multiplex reaction could beneglected. In addition, rare polyclonal TCRB V-J rearrangements wouldnot be mistaken for a clonal rearrangement even if they do not produce afully polyclonal Gaussian peak pattern, because PCR products of allpossible rearrangements are situated in the same size range.

For primer design, the rearrangeable pseudogenes or open reading framegenes with alterations in splicing sites, recombination signals and/orregulatory elements or changes of conserved amino acids were taken intoconsideration whenever possible. However, the main objective was tocover all functional Vβ genes. The priming efficiency of each Vβ primerwas checked for every Vβ gene using OLIGO 6.2 software. This led toprimers that were not strictly Vβ family specific and some of whichcovered Vβ gene segments of more than one family (FIG. 7B). Since the 13Jβ primers annealed to the same segment of each Jβ gene primer,dimerization made it necessary to split the J primers into two tubes.Initially, it was planned to use the primers in four sets of multiplexreactions as follows: all 23 Vβ primers in combination with the six Jβ1family primers (240-285 bp), all 23 Vβ primers with the seven Jβ2 familyprimers (240-285 bp), Dβ1 (280-320 bp) with the six Jβ1 primers, and Dβ1(280-320 bp) plus Dβ2 (170-210 bp) with the seven Jβ2 family primers.

Results of Initial Testing Phase

Initial monoplex testing of each possible primer combination was doneusing samples with known monoclonal TCRB rearrangements and polyclonalcontrols. PCR products of the expected size range were generated withdifferences in product intensity and signal profile for polyclonalsamples depending on the frequency of usage of distinct Vβ and Jβ genesegments. However, when the primers were combined in a multiplexreaction some Jβ2 rearrangements in particular were missed andnonspecific products in the tubes B and D were observed. In additioncross-priming between the Jβ1 and Jβ2 primers resulted in interpretationproblems. As a consequence the Jβ2 primers had to be redesigned and theprimer combinations in the different tubes had to be rearranged: Jβprimers Jβ2.2, 2.6 and 2.7 were slightly modified and added to tube A.The localization of the primers Jβ2.1, 2.3, 2.4 and 2.5 was shifted by 4bp downstream to avoid primer dimerization and cross priming with theremaining Jβ primers. Only nonspecific bands with varying intensityoutside the expected size range persisted in tube B (bands<150 bp, 221bp) and tube C (128 bp, 337 bp) using specific template DNA. However,because all nonspecific amplification products were outside the sizeranges of the TCRB specific products, they did not affect interpretationand were considered not to be a problem. However, using nonspecifictemplate controls one additional faint 273 bp aspecific peak in tube Awas visible by GS analysis. This product is completely suppressed whenthe DNA contains enough clonal or polyclonal TCRB rearrangements but canappear in samples comprising low numbers of lymphoid cells. In theinitial testing phase relatively faint V-D-J PCR products weregenerated. Thus we optimized PCR conditions for complete V-D-Jrearrangements by increasing MgCl₂ concentration and the amount of Taqpolymerase. Also usage of highly purified primers and application of ABIBuffer II instead of ABI Gold Buffer turned out to be very important.For detection of the incomplete Dβ-Jβ rearrangements, it was finallypossible to mix all Jβ primers into one tube without loss of sensitivityor information. Consequently, the total number of multiplex reactionscould be reduced to three tubes.

The finally approved primer set is (FIG. 7B):

tube A: 23 Vβ primers and 9 Jβ primers: Jβ1.1-1.6, 2.2, 2.6 and 2.7

tube B: 23 Vβ primers and 4 Jβ primers: Jβ2.1, 2.3, 2.4 and 2.5

tube C: Dβ1, Dβ2 and all 13 Jβ primers.

As tubes A and C contain Jβ1 and Jβ2 primers, differential labeling ofJβ1 and Jβ2 primers with different dyes (TET for Jβ1.1-1.6 and FAM forJβ2.1-2.7 primers) allows GS discrimination of Jβ1 or Jβ2 usage in tubeA and C reactions (see FIG. 13A).

Sensitivity testing was performed via dilution experiments with variouscell lines and patient samples with clonally rearranged TCRB genes inMNC. Single PCR dilution experiments generally reached sensitivitylevels of at least 0.1% to 1%. As expected, the sensitivity decreased inmultiplex testing,_probably due to an increase of backgroundamplification. Especially in GS analysis this background hamperedinterpretation due to the relative small length variation of the TCRBPCR products. Nevertheless, in 40 of 46 positive controls tested asensitivity of at least 1% to 10% was reached using heteroduplex orGeneScanning (Table 6).

Results of General Testing Phase

Eleven groups participated in the analysis of DNA from a series of 90Southern blot-defined malignant and reactive lymphoproliferativedisorders using the TCRB multiplex protocol. Every sample was analysedby HD in two laboratories and in six laboratories using GS analysis.Another three laboratories used both techniques for PCR analysis (FIGS.7C, D, and E). This testing phase as well as experience from use ofthese TCRB PCR assays raised some general issues about the protocol thatwere in part already described in the initial testing phase: 1. Thelimited length variation of the TCRB PCR products may hamper GSdetection of clonal signals within a polyclonal background. 2. Onlybands/peaks within the expected size range represent clonal TCRB generearrangements. Especially for tube A a nonspecific control DNA must beincluded to define the aspecific 273 bp peak that may occur insituations without competition. 3. It is extremely important to usehighly purified primers and ABI Buffer II (and not ABI Gold Buffer) forgood PCR results as well as the recommended PCR product preparationprotocol for HD analysis. Of the 90 Southern blot-defined casessubmitted, 29 were SB positive for monoclonal TCRB rearrangements. 25 ofthese clonal rearrangements (86%) were also detectable by the TCRB PCR.23 rearrangements were disclosed by GS and HD analysis, two additionalcases only by HD. One of the GS negative HD positive cases (FR-9) wasinterpreted as monoclonal on GS analysis by four of the ninelaboratories involved in the general testing phase (FIG. 7C). However,due to a significant polyclonal background, interpretation of the GSpatterns was difficult in this particular case. The other GS negative HDpositive case displayed an atypical PCR product in tube C with a size ofabout 400 bp (FIG. 7E). The PCR product was clearly visible in agarosegels and HD analysis but not by GS. After DNA sequencing of thisfragment a TCRB Dβ1-Dβ2 amplification product was identified explainingthe unlabelled PCR product. Four SB positive cases (NL-15, NL-16, GBN-2and FR-6) were neither detected by GS nor by HD analysis all of themwith an underlying B lymphoid malignancy. Possible explanations for thisfailure are atypical rearrangements (e.g. incomplete Vβ-Dβrearrangements),^(28, 104) sequence variations of the rearranged Vβ genesegments⁵¹ or a lack of sensitivity for particular rearrangements.

62 of the samples were considered to be polyclonal by SB. For 61 (98%)of these cases PCR results were concordant with at least one method ofanalysis, for 57 (92%) cases results were concordant using both methods.The one SB negative sample (ES-14) found to be monoclonal by HD and GSanalysis showed an incomplete Dβ2-rearrangement. For four samplesnon-uniform results were obtained: one sample was considered to beclonal by GS but only by 50% of the labs analyzing the PCR products byHD (GBS-4). Three samples were found to produce weak clonal signals onlyby HD analysis (ES-6, GBS-9 and DE-2). TCRB rearranged subclones beingtoo small to be detected by SB analysis may only be seen by the moresensitive PCR methodology. In B cell malignancies the detectedrearrangements may also represent clonal or oligoclonal expansions ofresidual T cells.¹⁰⁵ In this case these weak clonal PCR products shouldnot be regarded as evidence of a clonal T cell disorder. This stressesthe importance of the interpretation of the PCR results in context withother diagnostic tests and the clinical picture of the patients. OptimalPCR assessment of TCRB rearrangements is obtained by the combined use ofHD and GS analysis. Sensitivity may differ between the two detectionmethods as a function of clonal PCR product size compared to thepolyclonal size distribution: on the one hand HD analysis disperses thepolyclonal background from the clonal products and on the other hand PCRproducts outside the main size range allow a more sensitive GSdetection. Also the risk of false-positive results is reduced in thecombined use of HD and GS analysis. Furthermore, HD analysis allowsdetection of some additional atypical TCRB Dβ1-Dβ2 rearrangements thatcannot be detected by GS analysis of the PCR product as no labeledprimer is involved in amplification. However, GS analysis is in generalthe more informative method for samples with a high tumor load becausethe exact size of the monoclonal PCR product is indicated, which may beused for monitoring purposes and differentially labeled Jβ primersprovide additional information about JP gene usage.

Conclusion

In conclusion, the three-tube TCRB multiplex PCR system provides a newand convenient assay for clonality assessment in suspect T-cellproliferations with an unprecedentedly high clonality detection rate.

EXAMPLE 6 TCRG Gene Rearrangements Background

TCRG gene rearrangements have long been used for DNA PCR detection oflymphoid clonality and represent the “prototype” of restrictedrepertoire targets. It is a preferential target for clonality analysessince it is rearranged at an early stage of T lymphoid development,probably just after TCRD,¹⁰⁶ in both TCRαβ and TCRγδ lineage precursors.It is rearranged in greater than 90% of T-ALL, T-LGL and T-PLL, in50-75% of peripheral T-NHL and mycosis fungoides but not in true NK cellproliferations. It is also rearranged in a major part of B lineage ALLs,but much less so in B-NHL.^(1, 30, 73) Unlike several other Ig/TCR loci,the complete genomic structure has been known for many years. Itcontains a limited number of Vγ and Jγ segments. Amplification of allmajor Vγ-Jγ combinations is possible with limited number of four Vγ andthree Jγ primers.

The human TCRG locus on chromosome 7p14 contains 14 Vγ segments, onlyten of which have been shown to undergo rearrangement (FIG. 8A). Theexpressed Vγ repertoire includes only six Vγ genes (Vγ2, Vγ3, Vγ4, Vγ5,Vγ8 and Vγ9) but rearrangement also occurs with the ψVγ7, ψVγ10, γψVγ11segments.^(107, 108) Rearrangement of ψVγB (also known as Vγ12)¹⁰⁷ is soexceptional that it is rarely used in diagnostic PCR strategies.Rearranging Vγ segments can be subdivided into those belonging to theVγI family (VγfI: Vγ2, Vγ3, Vγ4, Vγ5, ψVγ7 and Vγ8; overallhomology >90% and highest between Vγ2 and Vγ4 and between Vγ3 and Vγ5)and the single member Vγ9, ψVγ10, ψVγ11 families. The TCRG locuscontains five Jγ segments: Jγ1.1 (JγP1), Jγ1.2 (JγP), Jγ1.3 (Jγ1), Jγ2.1(JγP2), Jγ2.3 (Jγ2), of which Jγ1.3 and Jγ2.3 are highly homologous, asare Jγ1.1 and Jγ2.1.¹⁰⁹

Whilst the restricted TCRG germline repertoire facilitates PCRamplification, the limited junctional diversity of TCRG rearrangementscomplicates distinction between clonal and polyclonal PCR products. TheTCRG locus does not contain D segments and demonstrates relativelylimited nucleotide additions. TCRG V-J junctional length thereforevaries by 20-30 bp, compared to approximately 60 bp for IGH and TCRD.The capacity to distinguish clonal from polyclonal TCRG rearrangementsdepends on the complexity of the polyclonal repertoire. In general,minor clonal populations using frequent Vγ-Jγ rearrangements such asVγfI-Jγ1.3/2.3 are at risk of being lost amidst the polyclonalrepertoire, whereas rare combinations will be detected with greatersensitivity. However, occasional polyclonal T lymphocytes demonstratingrare Vγ-Jγ rearrangements may be mistaken for a clonal rearrangement,due to absence of a polyclonal background for that type ofrearrangement. A further possible source of false positivity resultsfrom the presence of TCRγδ expressing T lymphocytes demonstrating“canonical” TCRG rearrangements, which do not demonstrate N nucleotideadditions. The most commonly recognized human canonical TCRGrearrangement involves the Vγ9-Jγ1.2 segments and occurs inapproximately 1% of blood T-lymphocytes.^(110, 111) It is thereforeextremely important to analyze TCRG PCR products using high resolutionelectrophoretic techniques or to separate PCR products on criteria otherthan purely on size, in order to reduce the risk of false positiveresults. It is also important to be aware of the profile of canonicalrearrangements and the situations in which they most commonly occur.Canonical Vγ9-Jγ1.2 rearrangements, for example, are found predominantlyin peripheral blood and increase in frequency with age, since theyresult from accumulation of TCRγδ⁺ T-lymphocytes.¹⁹

Unlike TCRD, TCRG is not deleted in TCRαβ lineage cells. Since TCRGrearrangements occur in both TCRαβ and TCRγδ lineage precursors, theiridentification cannot be used for determination of the type of T celllineage. Similarly, TCRG rearrangements occur in 60% of B lineageALLs,³⁰ implying that they can not be used for assessment of B vs. Tcell lineage in immature proliferations. However, they occur much lessfrequently in mature B lymphoproliferative disorders, including themajority of B-NHL,¹ and might therefore be used, in combination withclinical and immunophenotypic data, to determine lineage involvement inmature lymphoproliferative disorders.

The limited germline repertoire allows determination of Vγ and Jγsegment utilization, either by Southern blot or PCR analysis.Identification of Vγ and Jγ usage is not of purely academic interest,since specific amplification is required for MRD analysis.¹¹²

We undertook to develop a minimal number of multiplex TCRG strategieswhich would maintain optimal sensitivity and informativity, minimize therisk of false positive results and allow simple Vγ and Jγidentification, including by HD analysis or monofluorescent GSstrategies. We chose to include Vγ primers detecting all rearrangingsegments other than ψVγB (ψVγ12), given its rarity. In order to reducethe risk of falsely identifying canonical rearrangements as clonalproducts, we excluded the Jγ1.2 primer, since it is rarely involved inlymphoid neoplasms and is usually, although not always, associated witha TCRG rearrangement on the other allele.¹¹³

Primer Design

We initially developed 3Vγ and 2 Jγ primers, to be used in two multiplexreactions, as follows: one tube with Jγ1.3/2.3 with Vγ9 specific(160-190 bp), VγfI consensus (200-230 bp) and Vγ10/11 consensus (220-250bp) and a second tube with Jγ1.1/2.1 with Vγ9 specific (190-220 bp),VγfI consensus (230-260 bp) and Vγ10/11 consensus (250-280bp). Vγ usagewas to be identified by PCR product size by HD analysis. No distinctionbetween Jγ1.3 and Jγ2.3 or Jγ1.1 and Jγ2.1 was attempted.

Results of Initial Testing Phase

While all Vγ-Jγ combinations gave the expected profiles on single PCRamplification, multiplex amplification led to competition of larger PCRproducts, with preferential amplification of smaller fragments, andfailure to detect some VγfI and Vγ10/11 rearrangements. This was furthercomplicated by significant primer dimer formation between the Vγ10/11consensus and the VγfI primers. Competition between differently sizedfragments and primer dimer formation both led to unsatisfactorysensitivity and informativity and this strategy was therefore abandoned.

We reasoned that competition would be minimized by separating the mostfrequently used Vγ primers (VγfI and Vγ9) and combining them with Vγ10and Vγ11 specific primers, respectively. The latter rearrangements arerarely used and therefore minimize competition for the predominantrepertoires. The Vγ10/11 consensus primer was therefore replaced by twospecific Vγ primers which generated smaller PCR products (FIG. 8B). Bymixing Jγ1.3/2.3 and Jγ1.1/2.1 it was possible to maintain a two-tubemultiplex which allows approximate identification on the basis ofproduct size of Vγ usage by HD analysis and of both Jγ and Vγ usage byGS analysis.

The approved set of multiplex TCRG PCR tubes with four Vγ and two Jγprimers includes (FIG. 8B):

-   -   Tube A: VγfI+Vγ10+Jγ1.1/2.1+Jγ1.3/2.3    -   Tube B: Vγ9+Vγ11+Jγ1.1/2.1+Jγ1.3/2.3

The position and the sequence of the primers are shown in FIG. 8B. Theseprimers gave satisfactory amplification in both single and multiplex PCRformats and allowed detection of virtually all known Vγ-Jγ combinations.The competition of larger PCR fragments was no longer seen, although itcannot be excluded that some competition of Vγ9 or VγfI rearrangementsmay occur if these are present in a minority population.

Sensitivity of detection varied between 1% and 10%, as a function of thecomplexity of the polyclonal repertoire and the position of the clonalrearrangement relative to the polyclonal Gaussian peak.¹¹⁴Interpretation of ψVγ11 rearrangements can be difficult, since thenormal repertoire is extremely restricted and since these primitiverearrangements are often present in subclones.

Since the Vγ4 segment is approximately 40 bp longer than the other VγfImembers and Vγ4 rearrangements are relatively common in bothphysiological and pathological lymphoid cells, the polyclonal repertoirecan be skewed towards larger sized fragments, and clonal Vγ4-Jγ1.3/2.3rearrangements could theoretically be mistaken for VγfI-Jγ1.1/2.1rearrangements. The proximity of the different repertoires also makes Vγand Jγ identification much more reliable if differently labeled Jγprimers are used. For example, the use of a TET-labeled Jγ1.1/2.1 and aFAM labeled Jγ1.3/2.3 was tested in a single center and was shown togive satisfactory results (FIG. 13B). It is, however, possible toestimate Vγ and Jγ usage following GS analysis on the basis of sizealone (FIGS. 8C and D).

Results of General Testing Phase

Given the limited germline TCRG repertoire and the restricted junctionaldiversity, reactive T lymphocytes which have undergone TCRGrearrangements using a single Vγ and Jγ segment with variable CDR3sequences which are of uniform length, will migrate as an apparentclonal population by GS analysis. HD formation will disperse theserearrangements more easily and will therefore prevent their erroneousinterpretation as evidence of lymphoid clonality. In contrast, GSanalysis provides improved resolution and sensitivity compared to HDanalysis: For these reasons, optimal assessment of TCRG rearrangementsrequires both HD and GS analysis. If this is not possible, HD analysisalone is probably preferable, since it might be associated with a riskof false-negative results, whereas GS analysis alone will increase therisk of false-positive results.

Of the 18 TCRG rearrangements detected by Southern blotting in the 90cases, 16 were also detected by PCR. The minor Vγf1-Jγ1.3/2.3rearrangement detected by Southern in the NL-1 oligoclonal case, wasonly detected by PCR in a proportion of laboratories performing GSanalysis. A major Vγ9-Jγ1.3/2.3 rearrangement detected in GBS-6 wasfound to be polyclonal by both HD and GS in all laboratories and, assuch, probably represents a false-negative result.

Comparison of allele identification showed that, for all allelesidentified by Southern blotting, PCR Vγ and Jγ identification on thebasis of size gave concordant results. Seven rearrangements weredetected by Southern blotting but precise allele identification was notpossible. Six of these were clue to Jγ1.1/2.1 usage, suggesting that PCRallows preferential detection of this type of rearrangement.

Seventy two samples were considered to be polyclonal by Southern.Sixteen (22%) of these demonstrated a total of 24 rearrangements by TCRGPCR. Of these, 13 (81%) were B lymphoid proliferations. Sixteen of the24 clonal rearrangements were minor, with 15 only being detected by GSin the majority of laboratories. It is worth noting that, of these minorrearrangements, nine (39%) involved the ψVγ10 segment and eight (33%)Vγ9. ψVγ11 rearrangements were not detected. No ψVγ10 rearrangementswere detected by Southern blot analysis. PCR therefore allowed moresensitive detection of minor clonal ψVγ10 rearrangements, particularlyby GS analysis. It is likely that these rearrangements representresidual, predominantly TCRαβ lineage, T lymphocytes with a restrictedrepertoire, which may or may not be related to the underlying B lymphoidmalignancy. These minor peaks should obviously not be interpreted asevidence of a clonal T cell disorder. They emphasize the importance ofunderstanding the nature of TCRG rearrangements before using this locusas a PCR target in the lymphoid clonality diagnostic setting.Consequently, it is also extremely important to interpret TCRG generesults within their clinical context.

Conclusion

In conclusion, the two TCRG multiplex tubes allow detection of the vastmajority of clonal TCRG rearrangements. The potential risk of falsepositive results due to over-interpretation of minor clonal peaks can beminimized by the combined use of heteroduplex analysis and GeneScanningand by interpreting results within their clinical context, particularlywhen the apparent clonality involved the ψVγ10 and ψVγ11 segments. Therelative merits of TCRG compared to TCRB analysis for the detection ofclonal T lymphoproliferative disorders should be studied prospectively.They are likely to represent complementary strategies.

EXAMPLE 7 TCRD gene rearrangements: Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ, and Dδ-JδBackground

The human TCRD gene locus is located on chromosome 14q11.2 between theVα and Jα gene segments. The major part of the TCRD locus (Dδ-Jδ-Cδ) isflanked by TCRD-deleting elements, ψJα and δREC such that rearrangementof the deleting elements to each other or rearrangement of Vα to Jα genesegments causes deletion of the intermediate TCRD gene locus (FIG. 9A).The germline encoded TCRD locus consists of 8Vδ, 4Jδ, and 3Dδ genesegments, of which at least five of the eight Vδ gene segments can alsorearrange to Jα gene segments.¹¹⁵ Other Vα gene segments may also beutilized in TCRD gene rearrangements in rare cases. The WHO-IUISnomenclature¹¹⁰ for TCR gene segments uses a different numbering systemfor those V genes used mainly or exclusively in TCRδ chains from thosewhich can be used in either TCRα or TORδ chains. Thus TCRDV101S1 (Vδ1),TCRDV102S1 (Vδ2) and TCRDV103S1 (Vδ3) are used almost exclusively inTCRD rearrangements, whereas TCRADV6S1 (Vδ4), TCRADV21S1 (Vδ5) andTCRADV17S1 (Vδ6) can be used in either TCRδ or α chains. TCRADV28S1(Vδ7) and TCRADV14S1 (Vδ8) are used extremely rarely in TCRDrearrangements.

The germline-encoded repertoire of the TCRγδ⁺ T cells is small comparedto the TCRαβ⁺ T cells and the combinatorial repertoire is even morelimited due to preferential recombination in peripheral blood andthymocyte TCRγδ⁺ T cells. At birth, the repertoire of cord blood TCRγδ⁺T cells is broad, with no apparent restriction or preferred expressionof particular Vγ/Vδ combinations. During childhood, however, theperipheral blood TCRγδ⁺ T cell repertoire is strikingly shaped so thatVγ9/Vδ2 cells clearly dominate in adults.¹¹⁷ Studies have shown that Vδ1and Vδ2 repertoires become restricted with age leading to the appearanceof oligoclonal Vδ1⁺ and Vδ2⁺ cells in blood and intestine.¹¹⁸ TCRγδ⁺ Tcells are evenly distributed throughout human lymphoid tissues but thereis preferential expression of particular Vδ1 segments in specifiedanatomical localizations. Notably, most intraepithelial TCRγδ T cellsoccurring in the small intestine and in the colon express Vδ1.Similarly, Vδ1 is expressed by normal spleen TCRγδ⁺ T cells, but TCRγδ⁺T cells in the skin express the Vδ2 gene.

Although the small number of V, D and J gene segments available forrecombination limits the potential combinatorial diversity, the CDR3 orjunctional diversity is extensive due to the addition of N regions, Pregions and random deletion of nucleotides by recombinases. Thisdiversity is also extended by the recombination of up to three Dδsegments and therefore up to four N-regions within the rearranged TCRDlocus. This limited germline diversity encoded at the TCRD locus inconjunction with extensive junctional diversity results in a usefultarget for PCR analysis and TCRD recombination events have been usedmost extensively as clonal markers in both T and B cell acutelymphoblastic leukemia (ALL).^(119, 120) The TCRD locus is the first ofall TCR loci to rearrange during T cell ontogeny. The first event is aDδ2-Dδ3 rearrangement, followed by a Vδ2-(Dδ1-Dδ2)-Dδ3 rearrangement,and finally Vδ-Dδ-Jδ rearrangement. Immature rearrangements (Vδ2-Dδ3 orDδ2-Dδ3) occur in 70% of precursor B-ALL (and are therefore non lineagerestricted)” while there is a predominance of mature rearrangementscomprising incomplete Dδ2-Jδ1 and complete Vδ1, Vδ2, Vδ3 to Jδ1 found inT-ALL.^(23, 121) Thus specific primer sets can be used to identifydifferent types of complete and incomplete rearrangements correspondingto different types of ALL.¹²²

TCRγδ⁺ T-ALL form a relatively small subgroup of ALL, representing10-15% of T-ALL but still only constitute 2% of all ALL. Vδ1-Jδ1rearrangements predominate in TCRγδ⁺ T ALL; interestingly Vδ1 is neverfound in combination with Jδ segments other than Jδ1.^(15,20) Otherrecombinations occur in less than 25% of alleles. Furthermore,Vδ1-Jδ1-Cδ chains are almost always disulfide linked to either VγI orVγII gene families recombined to Jδ2.3-Cδ2. Such gene usage isconsistent with the immature thymic origin of these leukemic cells.

Most T cell lymphomas express TCRαβ while the minority express TCRγδ andcomprise of several distinct entities. Peripheral T cell lymphomas(PTCL) expressing TCRγδ comprise 8-13% of all PTCL and Vδ1-Jδ1 as wellas other Vδ to Jδ1 recombinations have beendocumented.^(123, 124)Hepatosplenic γδ T-cell lymphoma is derived fromsplenic TCRγδ T cells which normally express Vδ1. It is an uncommonentity that exhibits distinctive clinicopathologic features and geneusage analysis has indicated clonal Vδ1-Jδ1 rearrangements associatedwith these lymphomas.¹²⁵ Furthermore, the rare type of cutaneous TCRγδ⁺T cell lymphomas express Vδ2 and therefore appear to represent a clonalexpansion of TCRγδ⁺ T cells which normally reside in the skin.¹²⁸ Otherclonal TCRγδ proliferations include CD3⁺ TCRγδ⁺ large granularlymphocyte (LGL) proliferations which comprise about 5% of all CD3⁺ LGLand often show Vδ1-Jδ1 rearrangements.¹²⁷

The development of monoclonal antibodies towards framework regions ofTCRγδ and more recently to specific Vδ gene segments has helped identifyTCRγδ⁺ T cell populations by flow cytometric analysis,¹⁵ but PCRclonality studies are still required to identify whether thesepopulations represent clonal or polyclonal expansions.¹²⁸

Primer Design

The TCRD gene segments, consisting of eight Vδ, four Jδ and three Dδgene segments, show little or no homology to each other and sosegment-specific primers were designed which would not cross-anneal withother gene segments. Usage of Vδ7 and Vδ8 gene segments was consideredtoo rare to justify inclusion of primers for these segments and so,following the general guidelines according to the invention for primerdesign, a total of 16 primers were designed: 6 Vδ, 4 Jδ and 5′ and 3′ ofthe 3 DO gene segments (FIG. 9B). All primers were designed formultiplex together in any combination, but originally it was planned tohave one tube (A) with all V and all J primers which would amplify allthe complete V(D)J rearrangements and a second tube (B) with Vδ2,Dδ2-5′, Dδ3-3′ and Jδ1 primers to amplify the major partialrearrangements (Vδ2-Dδ3, Dδ2-Dδ3 and Dδ2-Jδ1). Together these tubesshould amplify 95% of known rearrangements. The other primers (Dδ1-5′,Dδ3-5′, Dδ1-3′ and Dδ2-3′) could be used to amplify other Dδ-Jδ, Vδ-Dδor Dδ-Dδ rearrangements, but were always intended to be optional.

Results of Initial Testing Phase

All primer pair combinations were tested using polyclonal DNA (tonsiland MNC). Most gave products of the expected size, but some (Dδ1-5′,Dδ1-3′ and Dδ2-3′) gave no visible product in combination with any otherprimer. Rearrangements involving these primer regions are likely to beextremely rare and so these, and Dδ3-5′, were excluded from subsequenttesting. Clonal cases for the six main rearrangements (Vδ1-Jδ1, Vδ2-Jδ1,Vδ3-Jδ1, Dδ2-Dδ3, Vδ2-Dδ3 and Dδ2-Jδ1) were tested initially in monoplexPCR and then in multiplex tubes A and B (see above). Serial dilutions ofclonal DNA in polyclonal DNA (tonsil or MNC) showed detectionsensitivities of at least 5% in all cases. However, in clonal cases withbiallelic rearrangements, which were clearly detected in single PCRreactions, the second, usually larger, allele often failed to amplify onmultiplexing. In addition, it was found, using a different set of clonalcases that several of the Vβ2-Jδ1 rearrangements failed to amplify. Apolymorphic site was subsequently identified at the position of theoriginal Vδ2 primer;¹²⁹ the frequency of this polymorphism in thegeneral population unknown, and so this primer was redesigned to a newregion of the Vδ2 gene segment, retested and found to amplify all cases.The problem with the failure to amplify the second allele was overcomeby increasing the MgCl₂ concentration from 1.5 mM to 2.0 mM.

We also tested the possibility of combining the two tubes into a singlemultiplex reaction. Twelve clonal cases were tested, which had a totalof 21 gene rearrangements between them. A single multiplex tubecontaining 12 primers (6 Vδ, 4 Jδ, Dδ2-5′ and Dδ3-3′) was used with ABIGold buffer and 2.0 mM MgCl₂to amplify all the cases. All generearrangements were indeed detected with a sensitivity of 0.5-10% by HDanalysis when diluted in polyclonal MNC DNA (Table 7). The only problemwith combining all TCRD primers in a single tube was the appearance of anonspecific band at about 90 bp in all amplifications, which was notpresent when the two separate multiplex tubes were used. Since the bandwas outside the size range of the TCRD products and did not interferewith interpretation, it was not considered to be a problem.

Results of General Testing Phase

The testing of the 90 Southern blot-defined samples in ten laboratoriesraised some general issues about the TCRD protocol:

Interpretation of some GS results was difficult. Because of the largesize range of products for the TCRD locus, there is no classicalGaussian distribution for polyclonal samples (see FIG. 9C) and this,coupled with the low usage of TCRD in many samples meant that in somecases it was hard to determine whether a sample was polyclonal orclonal. The same problem did not arise with HD analysis and so therecommendation is that GS should only be used for TCRD with extreme careand awareness of the potential problems.

The 90 bp nonspecific band was quite intense in some laboratories, butless so in others. It appeared to be weaker when using Buffer II ratherthan Gold buffer (confirmed by subsequent testing) and is also sensitiveto MgCl₂ concentration, becoming more intense as MgCl₂ concentrationincreases. This product has now been sequenced and found to be anunrelated gene utilizing the Dδ2 and Jδ3 primers.

The results of the general testing of the 90 Southern blot definedsamples showed that the overall concordance of all the PCR groups doingthe testing was very high (95%). Of the 90 cases, six were Southern blotpositive for TCRD clonal rearrangements, five of which were found to beclonal by PCR. The remaining case (DE-10, a T-ALL with high tumor load)was found to be polyclonal by all labs. Of the 84 Southern blot negativecases, 75 were found to be polyclonal by PCR, four were found to beclonal and the remaining five cases showed discordance between the GSand HD results. Of the clonal cases, two (DE-2 and GBS-9) were T-richB-NHLs with presumably low tumor load and so the results may reflect theincreased sensitivity of PCR over Southern blotting. The other twoclonal cases (GBS-15 and ES-7) had high tumor load. Of the five cases,which showed discrepancy between the GS and HD results, one (NL-1) was adifficult oligoclonal case, which caused problems for several otherloci. The remaining four were found to be polyclonal by HD and clonal byGS. In three of the cases (NL-13, NL-15 and NL-18) this may reflect thegreater sensitivity of GS over HD analysis, but the remaining case(PT-1, a reactive lymph node) may be attributed to “pseudoclonality” onGS analysis because of the limited repertoire of TCRD usage in somesamples.

Conclusion

In conclusion, the recommended protocol for detection of TCRD generearrangements is a single tube assay containing 12 primers fordetection of all major Vδ(D)Jδ, Vδ-Dδ, Dδ-Dδ and Dδ-Jδ rearrangementsusing Buffer II and 2.0 mM MgCl₂ to ensure maximum specificity anddetection. The preferred analysis method is HD, but GS may be used withdare if consideration is given to the problems of pseudoclonality causedby the limited usage of TCRD in some samples. However, the use ofmulti-color GeneScanning (see FIG. 13C) can be helpful in rapidrecognition of the different types of complete and incomplete TCRD generearrangements in the different types of ALL. With these limitations inmind, TCRD can nevertheless be a valuable target for the more immatureT-cell leukemias as well as TCR T-cell proliferations.

EXAMPLE 8 t(11;14) with BCL1-IGH Rearrangement Background

The t(11;14)(q13;q32) is characteristic for mantle cell lymphoma (MCL)because this cytogenetic reciprocal translocation was observed in 60-70%of MCL cases and only sporadically in other B-cell NHL.¹³⁰ Thebreakpoint region was originally cloned by Tsujimoto et al (1983) andreferred to as the BCL1-region.¹³¹ However in only few cases with acytogenetic t(11;14) a genomic breakpoint in the BCL1-region wasidentified. Using fiber and interphase FISH with probes covering theapproximately 750 kb 11q13-BCL1 region, in almost all MCL (33 out of 34)a breakpoint was observed and all breakpoints were confined to a regionof 360 kb 5′ of the cyclin D1 gene.^(132, 133) In nearly half of MCLcases (41%) the breakpoints were clustered within an 85 bp region thatwas referred to as the major translocation cluster region,BCL1-MTC.^(130, 134, 135) In most if not all cases of MCL the break atthe IGH locus located at 14q32 involves the J_(H) genes juxtaposing theIGH-Eμ enhancer to chromosome 11q13 sequences and consequently resultingin transcriptional activation of the cyclin D1 gene.¹³⁶ Cyclin D1together with CDK4 phosphorylates (and inactivates) pRB and allows forprogression through the G1 phase of the cell cycle. Because cyclin D1 issilent in B-lymphocytes and B-cell NHL other than MCL, and the presenceof this translocation correlates well with cyclin D1 expression, thisgene is considered to be the biological relevant target in MCL.¹³⁸ Bothexpression of cyclin D1 and/or the presence of t(11;14)(q13;q32) is usedas an additional tool in the differential diagnosis of NHL.² The goldstandard detection strategy for the presence of the t(11;14) that willidentify almost all breakpoints is interphase FISH usingbreakpoint-flanking probes in fresh or frozen material¹³³ as well as inarchival specimens.¹³⁷ However, a PCR based detection strategy for thet(11;14) might be useful for e.g. residual disease monitoring. Manygroups have developed PCR based assays to detect theBCL1/J_(H)-breakpoints, in general using a consensus J_(H)-primer incombination with primers in the BCL1-MTC region that were all located ina region of 392 bp.^(54, 55) Breaks within the BCL1-MTC region can occurup to 2 kb downstream of the MTC region, but the majority of breakpointsare tightly clustered within an 85 bp segment, immediately downstream ofthe reported most 3′-primer (“primer B” in^(54, 134)). Because breaks inthis BCL1-MTC-region account for only part of the breakpoints in the11q13-BCL1 region in MCL cases (41%), the PCR based strategy fort(11;14) seriously impairs the diagnostic capability with an high rateof false-negative results as compared to FISH.

The t(11;14)(q13;q32) has also been reported to be observed in otherB-cell proliferative diseases such as multiple myeloma (20%), SLVL(30%), B-PLL (33%) and B-CLL (⁸%)^(130, 138, 139) One reason for thepresence of the t(11;14) in B-CLL in some studies might be due to theincorrect classification of B-CLL.¹³⁰ In myeloma the breakpoints arequite different from those in MCL because (i) the frequency is muchlower; (ii) most breaks involve switch-class recombination sites; and(iii) although all tested cases are located in the same 360 kbBCL1-region there seems to be no preferential clustering within theBCL1-MTC region. On the other hand, in all cases with a break the cyclinD1 gene is activated. Of note, in a subgroup of multiple myelomas with aIGH-switch-break myeov, an additional region in the 11q13-BCL1 region,is involved.¹³⁸

Primer Design

Based on the location of the reported most-far 51-breakpoint andavailable nucleotide sequences from the BCL1-MTC region (GenBankaccession number S77049), we designed a single BCL1 primer(5′-GGATAAAGGCGAGGAGCATAA-3′) (SEQ ID NO:98) in the 472-bp region 5′ ofthis breakpoint by using the primer design program OL1GO6.2 relative tothe consensus J_(H) primer.

Results of Initial Testing Phase

Using the consensus JH-primer in combination with the singleBCL1-MTC-primer on a small series of MCL (n=5) previously identified aspositive with an in-house BCL1/JH-PCR using a similar consensusJH18-primer (18 nt) and 51-GCACTGTCTGGATGCACCGC-3′ (SEQ ID NO:131) asBCL1-MTC-primer, we initially compared both assays in parallel. Incontrast to the analysis of Ig/TCR gene rearrangements via GS and/or HDanalysis, the BCL1-J_(H) PCR products (as for BCL2-J_(H) products) areidentified via agarose gel electrophoresis using ethidium bromidestaining only. The results on the five positive arid two negativesamples were identical except that the PCR products were significantlyweaker. To evaluate whether we could increase the sensitivity of thePCR, we determined the effect of different concentrations of MgCl₂ andprimers, and different temperatures in a Stratagene-RobocyclerPCR-machine (all other PCR were done on ABI-480 or ABI-9700). Mostintriguing was the variation due to small changes in MgCl₂concentration. At 2.0 mM a weak nonspecific product of 550 bp becameapparent whereas at 2.5 mM and higher this nonspecific product was veryprominent in all DNAs including non-template DNA controls. At lowerconcentrations (less than 1.5 mM) no nonspecific fragments were observedbut the expected specific products were very weak. Hybridizations with aBCL1-MTC-internal oligo-probe (5′-ACCGAATATGCAGTGCAGC-3′) (SEQ IDNO:132) did not show hybridization to this 550 bp product. PCRs witheach of the primers separately revealed that the 550 bp product could begenerated by using the HI-consensus primer only. In some MCL cases, inaddition to the PCR-products ranging from 150-350 bp (FIG. 10B), largerspecific PCR-products might be apparent due to annealing of theconsensus JH-primer to downstream JH5 and JH6 segments as described forBCL2/JH.¹⁴⁰ From the initial testing phase the most optimalPCR-conditions for the BCL1-MTC/JH-PCR were: annealing temperature of60° C., 2.0 mM MgCl₂ and 10 pmol of each primer (for 35 PCR-cycles inthe ABI 9700).

To evaluate the specificity of the PCR on a larger series of cases, theBCL1-MTC/JH-PCR was performed in three laboratories on DNA from in total25 cases MCL that were all previously identified as positive within-house BCL1/JH-PCR, and from 18 negative controls. None of thenegative cases revealed a PCR-product whereas 22 of 25 positive casesshowed products of the expected size. In the three cases that did notreveal a product on agarose-gel, a product was detected with GSsuggesting that the sensitivity is lower when compared to in-house PCR.

The sensitivity of the PCR was evaluated by amplifying DNA dilutions ofa MCL in normal tonsillar DNA. A sensitivity between 10⁻³ and 10⁻⁴ wasobserved on agarose gel using the developed PCR-primers. An in-house PCRperformed in parallel on the same samples was at least 10× moresensitive. Hybridizations with the in-house BCL1-MTC-oligo-proberevealed a 10-100× higher sensitivity of both PCRs. Dilutions with DNAof an established cell line JVM2 (available through DSMZ;http://www.dsmz.de) with an BCL1-MTC/JH4-breakpoint⁵³ is used as ourstandard positive control. As a negative control normal tonsillar tissueor peripheral blood cells might be used, but almost any non-MCL B-cellNHL should be suitable because of the very low frequency of thisaberration.¹³⁰

Results of General Testing Phase

To evaluate inter-laboratory variations for the detection of breakpointsat the BCL1-MTC region, ten groups participated in the analysis of DNAfrom a series of 90 histologically defined malignant and reactivelymphoproliferations using the BCL1-MTC/JH-PCR protocol. All cases weredefined for their status at the Ig and TCR loci using Southernhybridization techniques. Of the 90 cases, seven were histologicallycharacterized as MCL. All seven MCL cases were shown to have a clonalIGH rearrangement by Southern hybridization. Assessment ofrearrangements within the BCL1-MTC-region at chromosome 11q13 by eitherSouthern hybridization or FISH was not performed in all cases. In six ofthe seven MCL cases the PCR-product was identified in all tenlaboratories. In MCL case NL-15 in six of the laboratories the expected1.8 kb PCR product was identified. This particular case carries anexceptional breakpoint with an uncommon large PCR-product (normallyranging from 150 to 350 bp) and represents the 3′-most-far detectableBCL1-MTC-breakpoint to our knowledge. In two of six labs the PCR productwas observed but initially considered as nonspecific because of itsuncommon size. In ES-4, characterized histologically as MCL in none ofthe ten labs a PCR-product could be detected suggesting that this casecarries a breakpoint outside the BCL1-MTC. It should be stressed thatthe MCL cases submitted to this series for the general testing phasewere selected and thus are expected to carry breaks at the BCL1-MTCregion at an higher incidence than normal. Importantly, except for onesingle case (FR-1), in all 83 other non-MCL cases including 16 casesthat were histologically characterized as B-CLL, no BCL1-MTC/JH-PCRproduct was detected in any laboratory. In case FR-1 histologicallycharacterized as B-CLL, in three of the ten labs a product wasidentified indicating that the number of cells with this break is low.The IGH status determined by Southern blot analysis revealed that thissample was composed of 90% clonal B-cells in good agreement with thehistological examination. PCR-based B-cell clonality analyses for IGHand IGK (sensitivity of approximately 1%) revealed a single clone andSouthern blot analysis for IGK showed a single major IGK rearrangementonly. In addition, Northern blot analysis for expression of cyclin D1did not show overexpression. All these data suggested that the verysmall number (less than 1%) t(11;14)-positive cells represent either (i)a subclone derived from the B-CLL, (ii) an independent secondB-malignancy or (iii) normal B-cells as described for t(14;18)-positiveB-cells in normal individuals.¹⁴⁰ However, with the available data ofthis patient at present we can not discriminate between these threealternatives. In summary, the analysis by the ten laboratoriesillustrates the high specificity of the BCL1-MTC/JH-PCR strategy.

To evaluate the presence of possible false-negative cases due to therelative low sensitivity of the PCR, in one laboratory the previouslydescribed in-house PCR (with about 10-fold higher sensitivity) wasperformed on DNA of all 90 cases and the PCR products of both assayswere also hybridized with an internal-BCL1-MTC oligo-probe thatincreases the sensitivity another 10-100-fold. This analysis revealed noPCR products in other cases.

Conclusion

We conclude that also the sensitivity of the BCL1-MTC/J_(H) PCR (between10⁻³ and 10⁻⁴) is sufficiently high for the detection of theBCL1-MTC/J_(H)-breakpoint in diagnostic material. The results of thisapproach are very encouraging and suggest that the definition of commonapproaches and reaction conditions can minimize erroneous results.However, it should be remembered that maximally about 50% of thet(11;14) breakpoints in MCL will be detected and that for diagnosisadditional detection tools are recommended.

EXAMPLE 9 t(14;18) with BCL2-IGH Rearrangement Background

The t(14;18) is one of the best characterized recurrent cytogeneticabnormalities in peripheral B cell lymphoproliferative disease.¹⁴¹ It isdetectable in up to 90% of follicular lymphomas and 20% of large cellB-cell lymphomas depending upon the diagnostic test used.¹⁴² As aconsequence of the translocation the BCL2 gene from 18q32 is placedunder the control of the strong enhancers of the IGH locus resulting inderegulation of its normal pattern of expression.^(143, 144) BCL2 islocated on the outer mitochondrial membrane and its normal function isto antagonize apoptosis and when deregulated it is intimately involvedin the pathogenesis of the tumor:¹⁴⁵⁻¹⁴⁸ As a consequence of this rolein pathogenesis the t(14;18) provides an ideal target for both diagnosisand molecular monitoring of residual disease.

The IGH locus is located at 14q32.3 with the V_(H) regions lyingtelomeric and the D_(H), J_(H) and constant regions placed morecentromeric. The transcriptional orientation is from telomere tocentromere with enhancers located 5′ of the V regions and between eachof the constant regions. The most common form of the translocationinvolves the process of VDJ recombination and one of the six germlineJ_(H) regions is closely opposed to BCL2. Most PCR based detectionstrategies have utilized a consensus J_(H) primer that will detect themajority of translocations.^(149, 150) In contrast to the IGH locus, thepattern of breaks in BCL2 is more complicated. BCL2 is located onchromosome 18q21 and is orientated 5′ to 3′ from centromere to telomere.The majority of breakpoints fall within the 150 bp MBR located in the 3′untranslated region of exon 3.¹⁵¹ As a consequence of the translocation,the Sμ enhancer located 3′ of the J_(H) regions is placed in closeproximity to the BCL2 gene leading to its deregulation. As moretranslocations have been investigated it has become apparent that thereare a number of other breakpoint regions which must be taken intoaccount for an efficient PCR detection strategy. Positioned 4 kbdownstream of the MBR is a further breakpoint region, the 3′MBRsubcluster, encompassing a region of 3.8 kb.¹⁵² The mer is located 20 kb3′ of the MBR and covers a region of 500 bp.¹⁵³ However, thoughanalogous to the MBR, the mer is more extensive than was initiallyenvisaged and a region 10 kb upstream of the mer, the 5′ mer subcluster,has been described.^(154, 155) In addition to these classicalbreakpoints a number of variant translocations are described where thebreaks occur 5′ of BCL2.¹⁵⁶ These are, however, rare and thus can not betaken into account using a PCR based detection strategy.

There is no single gold standard detection strategy for the t(14;18) anda combination of cytogenetics and Southern blotting have been generallyused.^(157, 158) Interphase FISH detection strategies offer anapplicable alternative that have the potential to pick up moretranslocations.¹⁵⁹ In contrast DNA based fiber FISH has been veryinformative for defining variant translocations but is unsuitable forroutine application.¹⁶⁰ For molecular diagnostic laboratories PCR baseddetection strategies offer rapid results, are generally applicable andcan be used for residual disease monitoring. However, the primerscommonly used have been derived on an ad hoc basis and have not beendesigned to take into account recent information on the molecularanatomy of the breakpoints. As a consequence when compared to goldstandard approaches, PCR based techniques only detect up to 60% oftranslocations which seriously impairs the diagnostic capability of PCR.Compounding this high percentage of false negative results is theproblem of false positive results arising from contamination from othersamples and previously amplified PCR products.

Primer Design

We initially evaluated a two tube multiplex system, one tube designed todetect breakpoints within the MBR and a second tube used to identifybreakpoints outside this region. The MBR strategy contained threeprimers MBR1, MBR2 and the consensus JH primer. The second multiplexreaction contained five primers, MCR1, MCR2, 5′mer, 3′ MBR1 and theconsensus J_(H) (FIG. 11A) and was designed to detect breakpoints withinthe mer, 5′mer and 3′ MBR regions.

Results of Initial Testing Phase

The evaluation of these primers was performed in three laboratories onDNA derived from a total of 124 cases of follicular lymphoma known tocarry a t(14;18). 109 cases (88%) were identified with an BCL2-IGHfusion, 83/124 (67%) were positive using the MBR multiplex and 26/124(21%) were positive using the non-MBR multiplex strategy. In 15/124(12%) cases there was no amplifiable PCR product. Further examination ofthe cases identified with the non-MBR multiplex showed that 11 (9%) hada breakpoint within the mer, five cases (4%) within the 5′mcr and 10/124(8%) within the 3′MBR.

To further investigate the value of this set of primers for thedetection of breakpoints within the 5′mer and 3′MBR sub-cluster regionsa series of 32 cases of t(14;18) positive follicular lymphomas known tobe germline at the MBR and mer by Southern hybridization were analyzedin one laboratory. Five of the cases had breakpoints within the 5′mer(260-490 bp) and were amplified using both the 5′mer primer in isolationand with the multiplex reaction. None of the remainder of cases showed apositive result. Of the series of 32 cases, nine were already known tohave breakpoints within the 3′MBR region and the multiplex approach wasable to detect 5/9 of these cases.

In order to improve the sensitivity of the assay within this region wedesigned three further primers that spanned the 3′MBR sub-clusterregion; 3′MBR2, 3′MBR3 and 3′MBR4 and combined them with 3′MBR1 and theconsensus JH in an additional multiplex reaction; 3′MBR multiplex (FIG.11). This new approach confirmed that eight of the 32 cases werepositive but missed the ninth case. The primers were then usedindividually and in this experiment 11 of the 32 cases were positive.The breakpoints were distributed as follows; 2/11 cases had a breakpointpresent between primer 3′MBR1 and 3′MBR2, 3/11 cases between primers3′MBR2 and 3′MBR3, 2/11 cases between primers 3′MBR3 and 3′MBR4 and theremaining four cases amplified using primer 3′MBR4 and were distributed200-1000 bp 3′ of this primer. In this series of cases there were threefalse negative results using the 3′MBR multiplex. One of the cases was atrue false negative where the break occurred in the middle of the 3′MBR,in proximity to an Alu repeat sequence. The translocation was detectedusing the 3′MBR3 primer when used in isolation and a product of 450 bpwas generated suggesting a reduced sensitivity of the multiplex. Theremaining two false negative cases generated products larger than 1000bp with the 3′MBR4 primer, placing them in the far 3′MBR not fullycovered by this approach. Further improvement in the sensitivity of the3′MBR assay has been achieved following the general testing phase ofthis study. Substituting primer 3′MBR3 with a new downstream primer 5′-GGTGACAGAGCAAAACATGAACA-3′ (see FIG. 11A) significantly improved boththe sensitivity and specificity of the 3′MBR assay.

Based on this, the 3′MBR multiplex was incorporated into our diagnosticstrategy. Analysis of the Southern blot defined cases was thereforecarried out using the three tube multiplex system presented in FIG. 11A.

Results of General Testing Phase

Inter-laboratory variations feature significantly in diagnostic PCRstrategies. To evaluate this, 11 groups participated in an extensiveexternal quality control exercise. DNA was extracted from a series of 90histologically defined malignant and reactive lymphoproliferations wereanalyzed using the t(14;18) multiplex protocol (FIGS. 11B, C, and D).All cases were defined for their status at the Ig and TCR loci usingSouthern hybridization techniques. Karyotypic confirmation of thet(14;18) was not available on this series. We therefore adopted anapproach requiring greater than 70% concordance between members of thenetwork for acceptance of the t(14;18). Of the 90 cases, 11 werecharacterized histologically as follicular lymphoma. All 11 cases wereshown to have a clonal IGH rearrangement by Southern hybridization.Assessment of rearrangements within the BCL2 gene was also performed bySouthern hybridization using specific probes to the MBR, mer and 3′MBRin 10/11 cases. 4/10 cases showed a rearrangement within the MBR thatwas concordant with the PCR result. A single case, GBS-7, shown to bemer multiplex positive, gave an inconclusive SB result with the merprobe. Immunophenotypically this case demonstrated two distinct clonalpopulations, representing approximately 5% and 15% of the originaldiagnostic material. The discrepancy between the two techniques in thiscase probably represents the reduced sensitivity of SB compared withPCR. There was no evidence of a 3′MBR rearrangement in any of theremaining cases by SB.

Of the six SB negative FCL cases, a single case; ES-7, showed a t(14;18)using the MBR multiplex. 5/11 FCL cases showed no evidence of a t(14;18)by either SB or PCR. A t(14;18) was detected in two further cases byPCR; FR-6, a case of DLBCL showed an MBR breakpoint and was identifiedby all 11 laboratories, this finding is compatible with previous studiesthat have detected a t(14;18) in 20-40% of DLBCL cases.^(161, 162) Usingthe 3′MBR multiplex, 10/11 laboratories reported a positive result forsample ES-12, this was a case of Hodgkin's disease which contained veryfew B cells. It is difficult to explain this result in the absence of anIGH rearrangement by Southern blotting. Contamination or incorrectlabeling of the sample at source is the most likely explanation.

Overall there was excellent concordance throughout the network, althoughsmall numbers of both false positive and false negative results wereencountered. Overall 12 false positive results were identified,representing less than 0.4% (12/3036) of the total number of analyses.These were reported by five laboratories and involved six of thesamples. The majority of the false positives (9/12) were found in threecases. Five false negative results, representing a 6% (5/88) failurerate, were reported by three laboratories, ES-7 was not detected by twolaboratories, three further groups within the network commented thatthis case had shown weak amplification signals with the MBR multiplex.The remaining three false negative cases were reported in isolation byindividual laboratories. The results of diagnoses using this approachare very encouraging and suggest that the definition of commonapproaches and reaction conditions can minimize erroneous results.

Conclusion

In conclusion, we have designed and evaluated a robust three-tubemultiplex PCR in order to maximize the detection of the t(14;18). Thisstrategy is capable of amplifying across the breakpoint region in themajority of cases of FCL with a cytogenetically defined translocation.Although the sensitivity of this strategy is lower than conventionalsingle round or nested PCR approaches, it is still perfectly acceptablefor diagnostic procedures. The widespread adoption of standardizedreagents and methodologies has helped to minimize inaccurate resultswithin this large multi-center network. However, it is noteworthy fromthe general testing phase of this study that it is impossible to detecta t(14;18) in all cases. This is certainly influenced by additionalmolecular mechanisms capable of deregulating the BCL2 gene.^(153, 164)

EXAMPLE 10 Use of DNA Extracted from Paraffin-Embedded Tissue Biopsiesand Development of Control Gene Primer Set Background

Fresh/frozen tissue is considered to be the ideal sample type forextraction of DNA for use in PCR-based clonality analysis. However,fresh/frozen material is not always available to diagnostic laboratoriesand in many laboratories throughout Europe, paraffin-embedded tissuesamples constitute the majority of diagnostic biopsies submitted foranalysis. DNA extracted from paraffin-embedded material is often of poorquality and so PCR protocols need to be evaluated for use with thesesample types before they can be widely used in diagnostic laboratories.

The integrity of DNA extracted from paraffin-embedded samples and itsamplification by PCR are affected by a number of factors such asthickness of tissue, fixative type, fixative time, length of storagebefore analysis, DNA extraction procedures and the co-extraction of PCRinhibitors.¹⁶⁵⁻¹⁷² Ten percent neutral buffered formalin (10% NBF) isthe most commonly used fixative, although laboratories also use a numberof other fixatives, including unbuffered formalin and Bouins. The use of10% NBF permits the amplification of DNA fragments of a wide range ofsizes whereas Bouins appears to be the least amenable for use in PCRanalysis.^(167, 168, 171, 173) The integrity of DNA fragments extractedfrom paraffin-embedded samples also depends on the length of time theblocks have been stored with the best results usually obtained fromblocks less than 2 years old, while blocks over 15 years old tend toyield very degraded fragments.'”

Primer Design

Initially, five pairs of control gene PCR primers were designed toamplify products of exactly 100, 200, 400, 600 and 1,000 bp in order toassess the quality of DNA submitted for analysis. The target genes wereselected on the basis of having large exons with open reading frames toreduce the risk of selecting polymorphic regions and the primers weredesigned for multiplex usage in the standardized protocols. Thefollowing target genes were selected: human thromboxane synthase gene(TBXAS1, Exon 9; GenBank Accession No D34621), human recombinationactivating gene (RAG1, Exon 2; GenBank Accession No M29474), humanpromyelocytic leukemia zinc finger gene (PLZF, Exon 1; GenBank AccessionNo AF060568), and human AF4 gene (Exon 3; GenBank Accession No 283679,and Exon 11; GenBank Accession No Z83687).

Results of Initial Testing Phase

The primer pairs were tested in separate reactions and subsequently inmultiple reactions using high molecular weight DNA. Due to the largesize range of the products (100 to 1,000 bp), it was necessary to varythe ratio of primer concentrations to obtain bands of equal intensitiesin the multiplex reactions. However, it proved extremely difficult to beable to amplify all the bands reproducibly and it was decided that the1,000 bp product was probably unnecessary, since all the PCR protocolsaccording to the invention give products of less than 600 bp. It wastherefore decided to exclude the 1,000 bp product in order to improvethe reproducibility of the assay. By increasing the MgCl₂ concentrationto 2 mM and adding the primers in a 1:1:1:2 ratio, it was possible toreproducibly amplify four bands (100, 200, 400 and 600 bp) of equalintensity from high molecular weight DNA samples. However, for DNAextracted from paraffin blocks, it was thought that an extra size markerat 300 bp would be extremely informative and that the 600 bp markermight not be necessary. Using the gene sequence for the 1,000 bp marker(PLZF), primers were redesigned to generate a 300 bp product. These weretested successfully both in monoplex reactions and in multiplexreactions combining the 100, 200, 300, 400 and 600 bp primers (see FIG.12A).

Thus two primer sets are available for assessing the quality of DNA foramplification: The 100, 200, 300 and 400 bp primers used at 2.5 pmoleach can be used for assessing DNA from paraffin-embedded tissues. Theaddition of the 600 bp primers at 5 pmol allows this set to be used tocheck the quality of any DNA sample for use with the primers andprotocols according to the invention. Both primer sets can be used withABI Buffer II and 2.0 mM MgCl₂ under standardized amplificationconditions. Products can be analyzed on 6% PAGE or 2% agarose (see FIG.12B).

Results of General Testing Phase

Forty five paraffin-embedded biopsies were collected corresponding to 30of the B-cell malignancies, eight of the T-cell malignancies and sevenof the reactive lymphoproliferations submitted as fresh/frozen tissuesamples. The age of the paraffin blocks as well as the methods offixation and embedding of the samples varied between National Networks.The ES samples were submitted as pre-cut sections, NL-14, 15 and 16 weresubmitted as DNA samples and the remaining biopsies were submitted asparaffin blocks. Five sections (10 μm each) were cut from the paraffinblocks and DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN)following the manufacturer's protocol for isolation of genomic DNA fromparaffin-embedded tissue. This method of DNA extraction was chosen sincethe kit can be used to rapidly extract good quality DNA from blood,fresh/frozen tissue and paraffin-embedded tissue and thus enables theparallel processing of a variety of sample types with assured qualitycontrol. Numerous protocols for extraction of DNA from paraffin-embeddedtissue for PCR analysis have been published.^(171, 172, 175-177) Many ofthese aim to reduce DNA degradation and co-extraction of PCR inhibitors,but many of these methods require prolonged extraction procedures andcan be unsuitable for use in the routine diagnosticlaboratory.^(166, 178, 179)

DNA sample concentration and integrity were estimated byspectrophotometry and by comparison of sample DNA with known standardson agarose gel electrophoresis. DNA samples (100 ng) were then analyzedfor integrity and amplifiability using the control gene PCR primers(100-400 bp) and assessed for clonality at all target loci using the PCRprotocols.

In the control gene PCR reaction of 24/45 cases the amplified productswere at least 300 bp, whereas in the remaining 21 samples the amplifiedproducts were 200 bp or less. No clear correlation between the qualityof the DNA and the age of the block or fixation method could bedemonstrated. Therefore it is likely that a combination of factors isresponsible for the DNA quality in these samples.

The DNA samples were evaluated for clonality using the 18 multiplex PCRreactions and were analyzed by both HD and GS. The number of paraffinsamples showing clonality and translocations at the nine target lociwere compared with the corresponding fresh/frozen sample data. Insamples with control gene PCR products of up to 200 bp, the overalldetection of clonality at the nine target loci was 9/55 (16%). Of the 46missed rearrangements, 45 could be explained by the fact that theexpected clonal PCR products had a molecular weight higher than themaximum size amplified by the sample in the control gene PCR. Theremaining sample (PT-9) amplified to 100 bp in the control gene PCR butthe expected 81 bp TCRG clonal product was not detected. In sampleswith'control gene PCR products of at least 300 bp, the overall detectionof clonality at the nine target loci was 42/55 (76%). Of the 13 missedrearrangements, five could again be explained by the fact that theexpected clonal PCR products were larger than the maximum size amplifiedby the sample in the control gene PCR. The remaining eight missedrearrangements could not be explained directly by the quality of theDNA. One false positive clonal result (GBN-9; IGL) was detected in areactive lymph node which may represent pseudoclonality.

PCR inhibitors are known to be present in DNA extracted from paraffinsamples. Dilution of the DNA sample may reduce the concentration ofthese inhibitors to levels that allow successful amplification to occur.To investigate the effect of diluting DNA samples on the efficiency ofamplification, four different concentrations of DNA were tested in thecontrol gene PCR reaction: 5, 50, 100 and 500 ng. We observed thatdilution of the DNA samples has a significant effect on the size of thePCR products in the control gene PCR. Overall, 24/45 cases (53%) showedan increased efficiency of amplification when diluted from 100 ng to 50ng. The optimal DNA concentration appears to be between 50 to 100 ngwhereas the use of 500 ng appears to inhibit the amplification of largeproducts (300 bp or above). Although the use of 5 ng of DNA givesacceptable results with the control gene PCR, this can lead to falsepositivity in PCR-based clonality assays due to the low representationof total lymphoid cell DNA.^(180, 181) More importantly, 5 ng of DNA hasno advantage over a dilution to 50 ng of DNA.

To assess whether the use of 50 ng of DNA would also increase thedetection of clonality, all the samples were retested at the IGH V-Jlocus using this DNA concentration. The number of clonal rearrangementsdetected in the three IGH V-J tubes using 100 ng of DNA was 12, comparedwith 23 using the corresponding fresh/frozen samples. The overalldetection of clonality at this locus increased to 17 out of 23 when 50ng of DNA was used, with an additional 9 FR1, 6 FR2 and 4 FR3 clonalproducts being detected. Thus dilution of the DNA can increase thedetection of clonal products, presumably because of dilution of PCRinhibitors. Logically, dilution of DNA is only likely to improve bothcontrol gene PCR results and the detection of clonality, if PCRinhibitors are present, not if the DNA sample is highly degraded.Therefore it is recommended that at least two dilutions of DNA aretested using the control gene PCR and that the dilution that gives thebetter result is used in subsequent clonality analysis.

Nine clonal rearrangements remained undetected after initial analysis,which could not be explained by DNA quality (TCRG in PT-9 and NL-11;TCRB in GBS-4; TCRD in NL-15; IGK in GBN-4, NL-4 and NL-5; IGHV-J_(H) inGBS-6 and GBS-8). These samples were retested using 50 ng of DNA, butonly one sample (GBS8; IGH) showed improved detection, suggesting thatother, unknown, factors can prevent amplification of specific targets ina small number of cases. However, it should be noted that for seven ofthese samples (NL-11, GBS-4, NL-15, GBN-4, NL-5, GBS-6 & GBS-8) clonalproducts were detected in at least one other locus. This demonstratesthat testing for clonality at multiple target loci increases thelikelihood of detecting clonal lymphocyte populations.

Conclusion

In conclusion, the protocols as provided herein work well with DNAextracted from paraffin-embedded material provided that the DNA canamplify products of 300 bp or more in the control gene PCR. Twoconcentrations of DNA are preferably tested in the control gene PCR andthe more ‘amplifiable’ concentration should be used in further testing,although with the proviso that concentrations of DNA less than 20 ng maycontribute to the detection of pseudoclonality due to the lowrepresentation of target lymphoid DNA.^(180, 181) Overall the data showthat assessment of DNA quality using the control gene PCR provides agood indication of the suitability of the DNA for clonality analysisusing the protocols provided. It is also important to note that thecontrol gene PCR will give no indication of the amount of lymphoid cellDNA present in the sample and therefore good quality DNA may stillproduce negative results for clonality analysis. To ensure monoclonalresults are reproducible (and to avoid potential pseudoclonality), allclonality assays, particularly using paraffin-extracted DNA, arepreferably performed in duplicate and analyzed by HD and GS, whereverpossible.

REFERENCES

-   1. Van Dongen J J M and Wolvers-Tettero I L M. Analysis of    immunoglobulin and T cell receptor genes. Part II: Possibilities and    limitations in the diagnosis and management of lymphoproliferative    diseases and related disorders. Clin Chim Acta 1991; 198: 93-174.-   2. Jaffe E S, Harris N L, Stein H, Vardiman J W, eds. World Health    Organization classification of tumours. Pathology and genetics of    tumours of haematopoietic and lymphoid tissues. 2001, IARC Press:    Lyon.-   3. Tonegawa S. Somatic generation of antibody diversity. Nature    1983; 302: 575-581.-   4. Davis M M and Bjorkman P J. T-cell antigen receptor genes and    T-cell recognition. Nature 1988; 334: 395-402.-   5. Van Dongen J J M, Szczepanski T, Adriaansen H J, Immunobiology of    leukemia, in Leukemia, E. S. Henderson, T. A. Lister, and M. F.    Greaves, Editors. 2002, WB Saunders Company: Philadelphia. p.    85-129.-   6. Szczepanski T, Pongers-Willemse M J, Langerak A W, van Dongen J    J M. Unusual immunoglobulin and T-cell receptor gene rearrangement    patterns in acute lymphoblastic leukemias. Curr Top Microbiol    Immunol 1999; 246: 205-215.-   7. Küppers R, Klein U, Hansmann M L, Rajewsky K. Cellular origin of    human B-cell lymphomas. N Engl J Med 1999; 341: 1520-1529.-   8. Smith B R, Weinberg D S, Robert N J, Towle M, Luther E, Pinkus G    S, Ault K A. Circulating monoclonal B lymphocytes in non-Hodgkin's    lymphoma. N Engl J Med 1984; 311: 1476-1481.-   9. Letwin B W, Wallace P K, Muirhead K A, Hensler G L, Kashatus W H,    Horan P K. An improved clonal excess assay using flow cytometry and    B-cell gating. Blood 1990; 75: 1178-1185.-   10. Fukushima P I, Nguyen P K, O'Grady P, Stetler-Stevenson M. Flow    cytometric analysis of kappa and lambda light chain expression in    evaluation of specimens for B-cell neoplasia. Cytometry 1996; 26:    243-252.-   11. McCoy J P, Jr., Overton W R, Schroeder K, Blumstein L, Donaldson    M H. Immunophenotypic analysis of the T cell receptor V beta    repertoire in CD4+ and CD8+ lymphocytes from normal peripheral    blood. Cytometry 1996; 26: 148-153.-   12. Van Dongen J J M, van den Beemd M W M, Schellekens M,    Wolvers-Tettero I L M, Langerak A W, Groeneveld K. Analysis of    malignant T cells with the Vβ antibody panel. Immunologist 1996; 4:    37-40.-   13. Van den Beemd M W M, Boor P P C, Van Lochem E G, Hop W C J,    Langerak A W, Wolvers-Tettero I L M, Hooijkaas H, Van Dongen J J M.    Flow ctometric analysis of the Vβ repertoire in healthy controls.    Cytometry 2000; 40: 336-345.-   14. Lima M, Almeida J, Santos A H, dos Anjos Teixeira M, Alguero M    C, Queiros M L, Balanzategui A, Justica B, Gonzalez M, San Miguel J    F, Orfao A. Immunophenotypic analysis of the TCR-Vbeta repertoire in    98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular    lymphocytes: utility in assessing clonality and insights into the    pathogenesis of the disease. Am J Pathol 2001; 159: 1861-1868.-   15. Langerak A W, Wolvers-Tettero I L M, van den Beemd M W M, van    Wering E R, Ludwig W-D, Hählen K, Necker A, van Dongen J J M.    Immunophenotypic and immunogenotypic characteristics of TCRgd⁺ T    cell acute lymphoblastic leukemia. Leukemia 1999; 13: 206-214.-   16. Langerak A W, van Den Beemd R, Wolvers-Tettero I L M, Boor P P,    van Lochem E G, Hooijkaas H, van Dongen J J M. Molecular and flow    cytometric analysis of the Vbeta repertoire for clonality assessment    in mature TCRalphabeta T-cell proliferations. Blood 2001; 98:    165-173.-   17. Semenzato G, Zambello R, Starkebaum G, Oshimi K, Loughran T P,    Jr. The lymphoproliferative disease of granular lymphocytes: updated    criteria for diagnosis. Blood 1997; 89: 256-260.-   18. Triebel F, Faure F, Graziani M, Jitsukawa S, Lefranc M P,    Hercend T. A unique V-J-C-rearranged gene encodes a gamma protein    expressed on the majority of CD3+ T cell    receptor-alpha/beta-circulating lymphocytes. J Exp Med 1988; 167:    694-699.-   19. Breit T M, Wolvers-Tettero I L, van Dongen J J. Unique selection    determinant in polyclonal V delta 2-J delta 1 junctional regions of    human peripheral gamma delta T lymphocytes. J Immunol 1994; 152:    2860-2864.-   20. Breit T M, Wolvers-Tettero I L M, Hählen K, Van Wering E R, Van    Dongen J J M. Limited combinatorial repertoire of gd T-cell    receptors expressed by T-cell acute lymphoblastic leukemias.    Leukemia 1991; 5: 116-124.-   21. Van Dongen J J M and Wolvers-Tettero I L M. Analysis of    immunoglobulin and T cell receptor genes. Part I: Basic and    technical aspects. Clin Chim Acta 1991; 198: 1-91.-   22. Beishuizen A, Verhoeven M A, Mol E J, Breit T M, Wolvers-Tettero    I L M, van Dongen J J M. Detection of immunoglobulin heavy-chain    gene rearrangements by Southern blot analysis: recommendations for    optimal results. Leukemia 1993; 7: 2045-2053.-   23. Breit T M, Wolvers-Tettero I L M, Beishuizen A, Verhoeven M-A J,    van Wering E R, van Dongen J J M. Southern blot patterns,    frequencies and junctional diversity of T-cell receptor d gene    rearrangements in acute lymphoblastic leukemia. Blood 1993; 82:    3063-3074.-   24. Beishuizen A, Verhoeven M A, Mol E J, van Dongen J J M.    Detection of immunoglobulin kappa light-chain gene rearrangement    patterns by Southern blot analysis. Leukemia 1994; 8: 2228-2236.-   25. Tumkaya T, Comans-Bitter W M, Verhoeven M A, van Dongen J J M.    Southern blot detection of immunoglobulin lambda light chain gene    rearrangements for clonality studies. Leukemia 1995; 9: 2127-2132.-   26. Tünakaya T, Beishuizen A, Wolvers-Tettero I L M, van Dongen J    J M. Identification of immunoglobulin lambda isotype gene    rearrangements by Southern blot analysis. Leukemia 1996; 10:    1834-1839.-   27. Moreau E J, Langerak A W, van Gastel-Mol E J, Wolvers-Tettero I    L M, Zhan M, Zhou Q, Koop B F, van Dongen J J M. Easy detection of    all T cell receptor gamma (TCRG) gene rearrangements by Southern    blot analysis: recommendations for optimal results. Leukemia 1999;    13: 1620-1626.-   28. Langerak A W, Wolvers-Tettero I L M, van Dongen J J M. Detection    of T cell receptor beta (TCRB) gene rearrangement patterns in T cell    malignancies by Southern blot analysis. Leukemia 1999; 13: 965-974.-   29. Hara J, Benedict S H, Mak T W, Gelfand E W. T cell receptor    alpha-chain gene rearrangements in B-precursor leukemia are in    contrast to the findings in T cell acute lymphoblastic leukemia.    Comparative study of T cell receptor gene rearrangement in childhood    leukemia. J Clin Invest 1987; 80: 1770-1777.-   30. Szczepanski T, Beishuizen A, Pongers-Willemse M J, Hahlen K, van    Wering E R, Wijkhuijs J M, Tibbe G J M, De Bruijn M A C, van Dongen    J J M. Cross-lineage T-cell receptor gene rearrangements occur in    more than ninety percent of childhood precursor-B-acute    lymphoblastic leukemias: alternative PCR targets for detection of    minimal residual disease. Leukemia 1999; 13: 196-205.-   31. Szczepanski T, Langerak A W, van Dongen J J, van Krieken J H.    Lymphoma with multi-gene rearrangement on the level of    immunoglobulin heavy chain, light chain, and T-cell receptor beta    chain. Am J Hematol 1998; 59: 99-100.-   32. Przybylski G, Oettle H, Ludwig W D, Siegert W, Schmidt C A.    Molecular characterization of illegitimate TCR delta gene    rearrangements in acute myeloid leukaemia. Br J Haematol 1994; 87:    301-307.-   33. Boeckx N, Willemse M J, Szczepanski T, van Der Velden V H J,    Langerak A W, Vandekerckhove P, van Dongen J J M. Fusion gene    transcripts and Ig/TCR gene rearrangements are complementary but    infrequent targets for PCR-based detection of minimal residual    disease in acute myeloid leukemia. Leukemia 2002; 16: 368-375.-   34. Szczepanski T, Pongers-Willemse M J, Langerak A W, Harts W A,    Wijkhuijs J M, van Wering E R, van Dongen J J M. Ig heavy chain gene    rearrangements in T-cell acute lymphoblastic leukemia exhibit    predominant D_(H)6-19 and D_(H)7-27 gene usage, can result in    complete V-D-J rearrangements, and are rare in T-cell receptor ab    lineage. Blood 1999; 93: 4079-4085.-   35. Kluin-Nelemans H C, Kester M G, van deCorput L, Boor P P,    Landegent J E, van Dongen J J, Willemze R, Falkenburg J H.    Correction of abnormal T-cell receptor repertoire during    interferon-alpha therapy in patients with hairy cell leukemia. Blood    1998; 91: 4224-4231.-   36. Sarzotti M, Patel D D, Li X, Ozaki D A, Cao S, Langdon S,    Parrott R E, Coyne K, Buckley R H. T cell repertoire development in    humans with SCID after nonablative allogeneic marrow    transplantation. J Immunol 2003; 170: 2711-2718.-   37. Mariani S, Coscia M, Even J, Peola S, Foglietta M, Boccadoro M,    Sbaiz L, Restagno G, Pileri A, Massaia M. Severe and long-lasting    disruption of T-cell receptor diversity in human myeloma after    high-dose chemotherapy and autologous peripheral blood progenitor    cell infusion. Br J Haematol 2001; 113: 1051-1059.-   38. Davis T H, Yockey C E, Balk S P. Detection of clonal    immunoglobulin gene rearrangements by polymerase chain reaction    amplification and single-strand conformational polymorphism    analysis. Am J Pathol 1993; 142: 1841-1847.-   39. Bourguin A, Tung R, Galili N, Sklar J. Rapid, nonradioactive    detection of clonal T-cell receptor gene rearrangements in lymphoid    neoplasms. Proc Natl Acad Sci USA 1990; 87: 8536-8540.-   40. Bottaro M, Berti E, Biondi A, Migone N, Crosti L. Heteroduplex    analysis of T-cell receptor gamma gene rearrangements for diagnosis    and monitoring of cutaneous T-cell lymphomas. Blood 1994; 83:    3271-3278.-   41. Langerak A W, Szczepanski T, van der Burg M, Wolvers-Tettero I L    M, van Dongen J J M. Heteroduplex PCR analysis of rearranged T cell    receptor genes for clonality assessment in suspect T cell    proliferations. Leukemia 1997; 11: 2192-2199.-   42. Kneba M, Bolz I, Linke B, Hiddemann W. Analysis of rearranged    T-cell receptor beta-chain genes by polymerase chain reaction (PCR)    DNA sequencing and automated high resolution PCR fragment analysis.    Blood 1995; 86: 3930-3937.-   43. Linke B, Bolz I, Fayyazi A, von Hofen M, Pott C, Bertram J,    Hiddemann W, Kneba M. Automated high resolution PCR fragment    analysis for identification of clonally rearranged immunoglobulin    heavy chain genes. Leukemia 1997; 11: 1055-1062.-   44. Szczepanski T, Flohr T, van der Velden V H, Bartram C R, van    Dongen J J. Molecular monitoring of residual disease using antigen    receptor genes in childhood acute lymphoblastic leukaemia. Best    Pract Res Clin Haematol 2002; 15: 37-57.-   45. Willemse M J, Seriu T, Hettinger K, d'Aniello E, Hop W C,    Panzer-Grumayer E R, Biondi A, Schrappe M, Kamps W A, Masera G,    Gadner H, Riehm H, Bartram C R, van Dongen J J. Detection of minimal    residual disease identifies differences in treatment response    between T-ALL and precursor B-ALL. Blood 2002; 99: 4386-4393.-   46. Lefranc MP. IMGT, the international ImMunoGeneTics database.    Nucleic Acids Res 2003; 31: 307-310.-   47. Lefranc M P. IMGT databases, web resources and tools for    immunoglobulin and T cell receptor sequence analysis,    http://imgt.cines.fr. Leukemia 2003; 17: 260-266.-   48. Ignatovich O, Tomlinson I M, Jones P T, Winter G. The creation    of diversity in the human immunoglobulin V(lambda) repertoire. J Mol    Biol 1997; 268: 69-77.-   49. Tümkaya T, van der Burg M, Garcia Sanz R, Gonzalez Diaz M,    Langerak A W, San Miguel J F, van Dongen J J M. Immunoglobulin    lambda isotype gene rearrangements in B-cell malignancies. Leukemia    2001; 15: 121-127.-   50. Arden B, Clark S P, Kabelitz D, Mak T W. Human T-cell receptor    variable gene segment families. Immunogenetics 1995; 42: 455-500.-   51. Rowen L, Koop B F, Hood L. The complete 685-kilobase DNA    sequence of the human beta T cell receptor locus. Science 1996; 272:    1755-1762.-   52. Quertermous T, Strauss W M, Van Dongen J J, Seidman J G. Human T    cell gamma chain joining regions and T cell development. J Immunol    1987; 138: 2687-2690.-   53. Rabbitts P, Douglas J, Fischer P, Nacheva E, Karpas A, Catovsky    D, Melo J, Baer R, Stinson M, Rabbitts T. Chromosome abnormalities    at 11q13 in B cell tumours. Oncogene 1988; 3: 99-103.-   54. Williams M E, Swerdlow S H, Meeker T C. Chromosome    t(11;14)(q13;q32) breakpoints in centrocytic lymphoma are highly    localized at the bcl-1 major translocation cluster. Leukemia 1993;    7: 1437-1440.-   55. Segal G H, Masih A S, Fox A C, Jorgensen T, Scott M, Braylan    R C. CD5-expressing B-cell non-Hodgkin's lymphomas with bcl-1 gene    rearrangement have a relatively homogeneous immunophenotype and are    associated with an overall poor prognosis. Blood 1995; 85:    1570-1579.-   56. Matsuda F, Ishii K, Bourvagnet P, Kuma K, Hayashida H, Miyata T,    Honjo T. The complete nucleotide sequence of the human    immunoglobulin heavy chain variable region locus. J Exp Med 1998;    188: 2151-2162.-   57. Ghia P, ten Boekel E, Rolink A G, Melchers F. B-cell    development: a comparison between mouse and man. Immunol Today 1998;    19: 480-485.-   58. Corbett S J, Tomlinson I M, Sonnhammer E L L, Buck D, Winter G.    Sequence of the human immunoglobulin diversity (D) segment locus: a    systematic analysis provides no evidence for the use of DIR    segments, inverted D segments, “minor” D segments or D-D    recombination. J Mol Biol 1997; 270: 587-597.-   59. Ichihara Y, Matsuoka H, Kurosawa Y. Organization of human    immunoglobulin heavy chain diversity gene loci. EMBO J 1988; 7:    4141-4150.-   60. Bertrand F E, III, Billips L G, Burrows P D, Gartland G L,    Kubagawa H, Schroeder H W, Jr. Ig D(H) gene segment transcription    and rearrangement before surface expression of the pan-B-cell marker    CD19 in normal human bone marrow. Blood 1997; 90: 736-744.-   61. Ghia P, ten Boekel E, Sanz E, de la Hera A, Rolink A,    Melchers F. Ordering of human bone marrow B lymphocyte precursors by    single-cell polymerase chain reaction analyses of the rearrangement    status of the immunoglobulin H and L chain gene loci. J Exp Med    1996; 184: 2217-2229.-   62. Szczepanski T, Willemse M J, van Wering E R, Weerden J F, Kamps    W A, van Dongen J J M. Precursor-B-ALL with DH-JH gene    rearrangements have an immature immunogenotype with a high frequency    of oligoclonality and hyperdiploidy of chromosome 14. Leukemia 2001;    15: 1415-1423.-   63. Davi F, Faili A, Gritti C, Blanc C, Laurent C, Sutton L, Schmitt    C, Merle-Beral H. Early onset of immunoglobulin heavy chain gene    rearrangements in normal human bone marrow CD34+ cells. Blood 1997;    90: 4014-4021.-   64. Szczepanski T, van't Veer M B, Wolvers-Tettero I L M, Langerak A    W, van Dongen J J M. Molecular features responsible for the absence    of immunoglobulin heavy chain protein synthesis in an IgH(-)    subgroup of multiple myeloma. Blood 2000; 96: 1087-1093.-   65. Schroeder H W, Jr. and Wang J Y. Preferential utilization of    conserved immunoglobulin heavy chain variable gene segments during    human fetal life. Proc Natl Acad Sci USA 1990; 87: 6146-6150.-   66. Raaphorst F M, Raman C S, Tami J, Fischbach M, Sanz I. Human Ig    heavy chain CDR3 regions in adult bone marrow pre-B cells display an    adult phenotype of diversity: evidence for structural selection of    D_(H) amino acid sequences. Int Immunol 1997; 9: 1503-1515.-   67. Lebecque S G and Gearhart P J. Boundaries of somatic mutation in    rearranged immunoglobulin genes: 5′ boundary is near the promoter,    and 3′ boundary is approximately 1 kb from V(D)J gene. J Exp Med    1990; 172: 1717-1727.-   68. Fukita Y, Jacobs H, Rajewsky K. Somatic hypermutation in the    heavy chain locus correlates with transcription. Immunity 1998; 9:    105-114.-   69. Zachau H G. The Immunologist 1996; 4: 49-54.-   70. Sellable K F and Zachau H G. The variable genes of the human    immunoglobulin kappa locus. Biol Chem Hoppe Seyler 1993; 374:    1001-1022.-   71. Weichhold G M, Ohnheiser R, Zachau H G. The human immunoglobulin    kappa locus consists of two copies that are organized in opposite    polarity. Genomics 1993; 16: 503-511.-   72. Siminovitch K A, Bakhshi A, Goldman P, Korsmeyer S J. A uniform    deleting element mediates the loss of kappa genes in human B cells.    Nature 1985; 316: 260-262.-   73. Szczepanski T, Langerak A W, Wolvers-Tettero I L M, Ossenkoppele    G J, Verhoef G, Stul M, Petersen E J, de Bruijn M A C, van't Veer M    B, van Dongen J J M. Immunoglobulin and T cell receptor gene    rearrangement patterns in acute lymphoblastic leukemia are less    mature in adults than in children: implications for selection of PCR    targets for detection of minimal residual disease. Leukemia 1998;    12: 1081-1088.-   74. Van der Velden V H J, Willemse M J, van der Schoot C E, van    Wering E R, van Dongen J J M. Immunoglobulin kappa deleting element    rearrangements in precursor-B acute lymphoblastic leukemia are    stable targets for detection of minimal residual disease by    real-time quantitative PCR. Leukemia 2002; 16: 928-936.-   75. van der Burg M, Tumkaya T, Boerma M, de Bruin-Versteeg S,    Langerak A W, van Dongen J J M. Ordered recombination of    immunoglobulin light chain genes occurs at the IGK locus but seems    less strict at the IGL locus. Blood 2001; 97: 1001-1008.-   76. Cannell P K, Amlot P, Attard M, Hoffbrand A V, Foroni L.    Variable kappa gene rearrangement in lymphoproliferative disorders:    an analysis of V kappa gene usage, VJ joining and somatic mutation.    Leukemia 1994; 8: 1139-1145.-   77. Frippiat J P, Williams S C, Tomlinson I M, Cook G P, Cherif D,    Le Paslier D, Collins J E, Dunham I, Winter G, Lefranc M P.    Organization of the human immunoglobulin lambda light-chain locus on    chromosome 22q11.2. Hum Mol Genet 1995; 4: 983-991.-   78. Williams S C, Frippiat J P, Tomlinson I M, Ignatovich O, Lefranc    M P, Winter G. Sequence and evolution of the human germline V lambda    repertoire. J Mol Biol 1996; 264: 220-232.-   79. Kawasaki K, Minoshima S, Nakato E, Shibuya K, Shintani A,    Schmeits J L, Wang J, Shimizu N. One-megabase sequence analysis of    the human immunoglobulin lambda gene locus. Genome Res 1997; 7:    250-261.-   80. Hieter P A, Korsmeyer S J, Waldmann T A, Leder P. Human    immunoglobulin kappa light-chain genes are deleted or rearranged in    lambda-producing B cells. Nature 1981; 290: 368-372.-   81. Vasicek T J and Leder P. Structure and expression of the human    immunoglobulin lambda genes. J Exp Med 1990; 172: 609-620.-   82. Taub R A, Hollis G F, Hieter P A, Korsmeyer S, Waldmann T A,    Leder P. Variable amplification of immunoglobulin lambda light-chain    genes in human populations. Nature 1983; 304: 172-174.-   83. van der Burg M, Barendregt B H, van Gastel-Mol E J, Tumkaya T,    Langerak A W, van Dongen J J. Unraveling of the polymorphic C lambda    2-C lambda 3 amplification and the Ke+Oz-polymorphism in the human    Ig lambda locus. J Immunol 2002; 169: 271-276.-   84. Bridges S L, Jr. Frequent N addition and clonal relatedness    among immunoglobulin lambda light chains expressed in rheumatoid    arthritis synovia and PBL, and the influence of V lambda gene    segment utilization on CDR3 length. Mol Med 1998; 4: 525-553.-   85. Kiyoi H, Naito K, Ohno R, Saito H, Naoe T. Characterization of    the immunoglobulin light chain variable region gene expressed in    multiple myeloma. Leukemia 1998; 12: 601-609.-   86. Farner N L, Dorner T, Lipsky P E. Molecular mechanisms and    selection influence the generation of the human V lambda J lambda    repertoire. J Immunol 1999; 162: 2137-2145.-   87. Ignatovich O, Tomlinson I M, Popov A V, Bruggemann M, Winter G.    Dominance of intrinsic genetic factors in shaping the human    immunoglobulin Vlambda repertoire. J Mol Biol 1999; 294: 457-465.-   88. Wei S, Charmley P, Robinson M A, Concannon P. The extent of the    human germline T-cell receptor V beta gene segment repertoire.    Immunogenetics 1994; 40: 27-36.-   89. Charmley P, Wei S, Concannon P. Polymorphisms in the TCRB-V2    gene segments localize the Tcrb orphon genes to human chromosome    9p21. Immunogenetics 1993; 38: 283-286.-   90. Robinson M A, Mitchell M P, Wei S, Day C E, Zhao T M,    Concannon P. Organization of human T-cell receptor beta-chain genes:    clusters of V beta genes are present on chromosomes 7 and 9. Proc    Natl Acad Sci USA 1993; 90: 2433-2437.-   91. Toyonaga B, Yoshikai Y, Vadasz V, Chin B, Mak T W. Organization    and sequences of the diversity, joining, and constant region genes    of the human T-cell receptor beta chain. Proc Natl Acad Sci USA    1985; 82: 8624-8628.-   92. Liu D, Callahan J P, Dau P C. Intrafamily fragment analysis of    the T cell receptor beta chain CDR3 region. J Immunol Methods 1995;    187: 139-150.-   93. Tsuda S, Rieke S, Hashimoto Y, Nakauchi H, Takahama Y. 11-7    supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal    liver progenitor cells. J Immunol 1996; 156: 3233-3242.-   94. Weidmann E, Whiteside T L, Giorda R, Herberman R B, Trucco M.    The T-cell receptor V beta gene usage in tumor-infiltrating    lymphocytes and blood of patients with hepatocellular carcinoma.    Cancer Res 1992; 52: 5913-5920.-   95. Jores R and Meo T. Few V gene segments dominate the T cell    receptor beta-chain repertoire of the human thymus. J Immunol 1993;    151: 6110-6122.-   96. Rosenberg W M, Moss P A, Bell J I. Variation in human T cell    receptor V beta and J beta repertoire: analysis using anchor    polymerase chain reaction. Eur J Immunol 1992; 22: 541-549.-   97. Pongers-Willemse M J, Seriu T, Stolz F, d'Aniello E, Gameiro P,    Pisa P, Gonzalez M, Bartram C R, Panzer-Grumayer E R, Biondi A, San    Miguel J F, van Dongen J J M. Primers and protocols for standardized    MRD detection in ALL using immunoglobulin and T cell receptor gene    rearrangements and TAL1 deletions as PCR targets. Report of the    BIOMED-1 Concerted Action: Investigation of minimal residual disease    in acute leukemia. Leukemia 1999; 13: 110-118.-   98. Hansen-Hagge T E, Yokota S, Bartram C R. Detection of minimal    residual disease in acute lymphoblastic leukemia by in vitro    amplification of rearranged T-cell receptor delta chain sequences.    Blood 1989; 74: 1762-1767.-   99. Cave H, Guidal C, Rohrlich P, Delfau M H, Broyart A, Lescoeur B,    Rahimy C, Fenneteau O, Monplaisir N, d'Auriol L, Elion J, Vilmer E,    Grandchamp B. Prospective monitoring and quantitation of residual    blasts in childhood acute lymphoblastic leukemia by polymerase chain    reaction study of delta and gamma T-cell receptor genes. Blood 1994;    83: 1892-1902.-   100. Gorski J, Yassai M, Zhu X, Kissela B, Kissella B, Keever C,    Flomenberg N. Circulating T cell repertoire complexity in normal    individuals and bone marrow recipients analyzed by CDR3 size    spectratyping. Correlation with immune status. J Immunol 1994; 152:    5109-1519.-   101. McCarthy K P, Sloane J P, Kabarowski J H, Matutes E, Wiedemann    L M. The rapid detection of clonal T-cell proliferations in patients    with lymphoid disorders. Am J Pathol 1991; 138: 821-828.-   102. Assaf C, Hummel M, Dippel E, Goerdt S, Muller H H,    Anagnostopoulos I, Orfanos C E, Stein H. High detection rate of    T-cell receptor beta chain rearrangements in T-cell    lymphoproliferations by family specific polymerase chain reaction in    combination with the GeneScan technique and DNA sequencing. Blood    2000; 96: 640-646.-   103. O'Shea U, Wyatt J I, Howdle P D. Analysis of T cell receptor    beta chain CDR3 size using RNA extracted from formalin fixed    paraffin wax embedded tissue. J Clin Pathol 1997; 50: 811-814.-   104. Duby A D and Seidman J G. Abnormal recombination products    result from aberrant DNA rearrangement of the human T-cell antigen    receptor beta-chain gene. Proc Natl Acad Sci USA 1986; 83:    4890-4894.-   105. Alatrakchi N, Farace F, Frau E, Carde P, Munck J N, Triebel F.    T-cell clonal expansion in patients with B-cell lymphoproliferative    disorders. J Immunother 1998; 21: 363-370.-   106. Blom B, Verschuren M C, Heemskerk M H, Bakker A Q, van    Gastel-Mol E J, Wolvers-Tettero I L, van Dongen J J M, Spits H. TCR    gene rearrangements and expression of the pre-T cell receptor    complex during human T-cell differentiation. Blood 1999; 93:    3033-3043.-   107. Chen Z, Font M P, Loiseau P, Bories J C, Degos L, Lefranc M P,    Sigaux F. The human T-cell V gamma gene locus: cloning of new    segments and study of V gamma rearrangements in neoplastic T and B    cells. Blood 1988; 72: 776-783.-   108. Zhang X M, Tonnelle C, Lefranc M P, Huck S. T cell receptor    gamma cDNA in human fetal liver and thymus: variable regions of    gamma chains are restricted to V gamma I or V9, due to the absence    of splicing of the V10 and V11 leader intron. Eur J Immunol 1994;    24: 571-578.-   109. Huck S and Lefranc M P. Rearrangements to the JP1, JP and JP2    segments in the human T-cell rearranging gamma gene (TRG gamma)    locus. FEBS Lett 1987; 224: 291-296.-   110. Delfau M H, Hance A J, Lecossier D, Vilmer E, Grandchamp B.    Restricted diversity of V gamma 9-JP rearrangements in unstimulated    human gamma/delta T lymphocytes. Eur J Immunol 1992; 22: 2437-2443.-   111. Porcelli S, Brenner M B, Band H. Biology of the human gamma    delta T-cell receptor. Immunol Rev 1991; 120: 137-183.-   112. Van der Velden V H J, Wijkhuijs J M, Jacobs D C H, van Wering E    R, van Dongen J J M. T cell receptor gamma gene rearrangements as    targets for detection of minimal residual disease in acute    lymphoblastic leukemia by real-time quantitative PCR analysis.    Leukemia 2002; 16: 1372-1380.-   113. Szczepanski T, Langerak A W, Willemse M J, Wolvers-Tettero I L    M, van Wering E R, van Dongen J J M. T cell receptor gamma (TCRG)    gene rearrangements in T cell acute lymphoblastic leukemia reflect    “end-stage” recombinations: implications for minimal residual    disease monitoring. Leukemia 2000; 14: 1208-1214.-   114. Delabesse E, Burtin M L, Millien C, Madonik A, Arnulf B,    Beldjord K, Valensi F, Macintyre E A. Rapid, multifluorescent TCRG    Vgamma and Jgamma typing: application to T cell acute lymphoblastic    leukemia and to the detection of minor clonal populations. Leukemia    2000; 14: 1143-1152.-   115. Verschuren M C, Wolvers-Tettero I L, Breit T M, van Dongen J J.    T-cell receptor V delta-J alpha rearrangements in human thymocytes:    the role of V delta-J alpha rearrangements in T-cell receptor-delta    gene deletion. Immunology 1998; 93: 208-212.-   116. Nomenclature for T-cell receptor (TCR) gene segments of the    immune system. WHO-IUIS Nomenclature Sub-Committee on TCR    Designation. Immunogenetics 1995; 42: 451-453.-   117. Kabelitz D, Wesch D, Hinz T. Gamma delta T cells, their T cell    receptor usage and role in human diseases. Springer Semin    Immunopathol 1999; 21: 55-75.-   118. Shen J, Andrews D M, Pandolfi F, Boyle L A, Kersten C M,    Blatman R N, Kurnick J T. Oligoclonality of Vdelta1 and Vdelta2    cells in human peripheral blood mononuclear cells: TCR selection is    not altered by stimulation with gram-negative bacteria. J Immunol    1998; 160: 3048-3055.-   119. Breit T M, Wolvers-Tettero I L M, Hählen K, Van Wering E R, Van    Dongen J J M. Extensive junctional diversity of gd T-cell receptors    expressed by T-cell acute lymphoblastic leukemias: implications for    the detection of minimal residual disease. Leukemia 1991; 5:    1076-1086.-   120. Langlands K, Eden O B, Micallef-Eynaud P, Parker A C, Anthony    R S. Direct sequence analysis of TCR V delta 2-D delta 3    rearrangements in common acute lymphoblastic leukaemia and    application to detection of minimal residual disease. Br J Haematol    1993; 84: 648-655.-   121. Schneider M, Panzer S, Stolz F, Fischer S, Gadner H,    Panzer-Grumayer E R. Crosslineage TCR delta rearrangements occur    shortly after the DJ joinings of the IgH genes in childhood    precursor B ALL and display age-specific characteristics. Br J    Haematol 1997; 99: 115-121.-   122. Hettinger K, Fischer S, Panzer S, Panzer-Grumayer E R.    Multiplex PCR for TCR delta rearrangements: a rapid and specific    approach for the detection and identification of immature and mature    rearrangements in ALL. Br J Haematol 1998; 102: 1050-1054.-   123. Theodorou I, Raphael M, Bigorgne C, Fourcade C, Lahet C, Cochet    G, Lefranc M P, Gaulard P, Farcet J P. Recombination pattern of the    TCR gamma locus in human peripheral T-cell lymphomas. J Pathol 1994;    174: 233-242.-   124. Kanavaros P, Farcet J P, Gaulard P, Haioun C, Divine M, Le    Couedic J P, Lefranc M P, Reyes F. Recombinative events of the T    cell antigen receptor delta gene in peripheral T cell lymphomas. J    Clin Invest 1991; 87: 666-672.-   125. Przybylski G K, Wu H, Macon W R, Finan J, Leonard D G, Felgar R    E, DiGiuseppe J A, Nowell P C, Swerdlow S H, Kadin M E, Wasik M A,    Salhany K E. Hepatosplenic and subcutaneous panniculitis-like    gamma/delta T cell lymphomas are derived from different Vdelta    subsets of gamma/delta T lymphocytes. J Mol Diagn 2000; 2: 11-19.-   126. Kadin M E. Cutaneous gamma delta T-cell lymphomas—how and why    should they be recognized? Arch Dermatol 2000; 136: 1052-1054.-   127. Hodges E, Quin C, Farrell A M, Christmas S, Sewell H F, Doherty    M, Powell R J, Smith J L. Arthropathy, leucopenia and recurrent    infection associated with a TcR gamma delta population. Br J    Rheumatol 1995; 34: 978-983.-   128. Van Oostveen J W, Breit T M, de Wolf J T, Brandt R M, Smit J W,    van Dongen J J M, Borst J, Melief C J. Polyclonal expansion of    T-cell receptor-gd+ T lymphocytes associated with neutropenia and    thrombocytopenia. Leukemia 1992; 6: 410-418.-   129. Triebel F, Faure F, Mami-Chouaib F, Jitsukawa S, Griscelli A,    Genevee C, Roman-Roman S, Hercend T. A novel human V delta gene    expressed predominantly in the Ti gamma A fraction of gamma/delta+    peripheral lymphocytes. Eur J Immunol 1988; 18: 2021-2027.-   130. De Boer C J, van Krieken J H, Schuuring E, Kluin P M.    Bcl-1/cyclin D1 in malignant lymphoma. Ann Oncol 1997; 8: 109-117.-   131. Tsujimoto Y, Yunis J, Onorato-Showe L, Erikson J, Nowell P C,    Croce C M. Molecular cloning of the chromosomal breakpoint of B-cell    lymphomas and leukemias with the t(11;14) chromosome translocation.    Science 1984; 224: 1403-1406.-   132. Vaandrager J W, Kleiverda J K, Schuuring E, Kluin-Nelemans J C,    Raap A K, Kluin P M. Cytogenetics on released DNA fibers. Verh Dtsch    Ges Pathol 1997; 81: 306-311.-   133. Vaandrager J W, Schuuring E, Zwikstra E, de Boer C J, Kleiverda    K K, van Krieken J H, Kluin-Nelemans H C, van Ommen G J, Raap A K,    Kluin P M. Direct visualization of dispersed 11q13 chromosomal    translocations in mantle cell lymphoma by multicolor DNA fiber    fluorescence in situ hybridization. Blood 1996; 88: 1177-1182.-   134. Pott C, Tiemann M, Linke B, Ott M M, von Hofen M, Bolz I,    Hiddemann W, Parwaresch R, Kneba M. Structure of Bcl-1 and IgH-CDR3    rearrangements as clonal markers in mantle cell lymphomas. Leukemia    1998; 12: 1630-1637.-   135. Luthra R, Hai S, Pugh W C. Polymerase chain reaction detection    of the t(11;14) translocation involving the bcl-1 major    translocation cluster in mantle cell lymphoma. Diagn Mol Pathol    1995; 4: 4-7.-   136. de Boer C J, Schuuring E, Dreef E, Peters G, Bartek J, Kluin P    M, van Krieken J H. Cyclin D1 protein analysis in the diagnosis of    mantle cell lymphoma. Blood 1995; 86: 2715-2723.-   137. Haralambieva E, Kleiverda K, Mason D Y, Schuuring E, Kluin P M.    Detection of three common translocation breakpoints in non-Hodgkin's    lymphomas by fluorescence in situ hybridization on routine    paraffin-embedded tissue sections. J Pathol 2002; 198: 163-170.-   138. Janssen J W, Vaandrager J W, Heuser T, Jauch A, Kluin P M,    Geelen E, Bergsagel P L, Kuehl W M, Drexler H G, Otsuki T, Bartram C    R, Schuuring E. Concurrent activation of a novel putative    transforming gene, myeov, and. cyclin D1 in a subset of multiple    myeloma cell lines with t(11;14)(q13;q32). Blood 2000; 95:    2691-2698.-   139. Troussard X, Mauvieux L, Radford-Weiss I, Rack K, Valensi F,    Garand R, Vekemans M, Flandrin G, Macintyre E A. Genetic analysis of    splenic lymphoma with villous lymphocytes: a Groupe Francais    d'Hematologie Cellulaire (GFHC) study. Br J Haematol 1998; 101:    712-721.-   140. Limpens J, Stad R, Vos C, de Vlaam C, de Jong D, van Ommen G J,    Schuuring E, Kluin P M. Lymphoma-associated translocation t(14;18)    in blood. B cells of normal individuals. Blood 1995; 85: 2528-2536.-   141. Fukuhara S, Rowley J D, Variakojis D, Golomb H M. Chromosome    abnormalities in poorly differentiated lymphocytic lymphoma. Cancer    Res 1979; 39: 3119-3128.-   142. Weiss L M, Warnke R A, Sklar J, Cleary M L. Molecular analysis    of the t(14;18) chromosomal translocation in malignant lymphomas. N    Engl J Med 1987; 317: 1185-1189.-   143. Bakhshi A, Jensen J P, Goldman P, Wright J J, McBride O W,    Epstein A L, Korsmeyer S J. Cloning the chromosomal breakpoint of    t(14;18) human lymphomas: clustering around JH on chromosome 14 and    near a transcriptional unit on 18. Cell 1985; 41: 899-906.-   144. Cleary M L and Sklar J. Nucleotide sequence of a t(14;18)    chromosomal breakpoint in follicular lymphoma and demonstration of a    breakpoint-cluster region near a transcriptionally active locus on    chromosome 18. Proc Natl Acad Sci USA 1985; 82: 7439-7443.-   145. Korsmeyer S J. BCL-2 gene family and the regulation of    programmed cell death. Cancer Res 1999; 59: 1693s-1700s.-   146. Lithgow T, van Driel R, Bertram J F, Strasser A. The protein    product of the oncogene bcl-2 is a component of the nuclear    envelope, the endoplasmic reticulum, and the outer mitochondrial    membrane. Cell Growth Differ 1994; 5: 411-417.-   147. Woodland R T, Schmidt M R, Korsmeyer S J, Gravel K A.    Regulation of B cell survival in xid mice by the proto-oncogene    bcl-2. J Immunol 1996; 156: 2143-2154.-   148. Hsu S Y, Lai R J, Finegold M, Hsueh A J. Targeted    overexpression of Bcl-2 in ovaries of transgenic mice leads to    decreased follicle apoptosis, enhanced folliculogenesis, and    increased germ cell tumorigenesis. Endocrinology 1996; 137:    4837-4843.-   149. Lee M S, Chang K S, Cabanillas F, Freireich E J, Trujillo J M,    Stass S A. Detection of minimal residual cells carrying the t(14;18)    by DNA sequence amplification. Science 1987; 237: 175-178.-   150. Crescenzi M, Seto M, Herzig G P, Weiss P D, Griffith R C,    Korsmeyer S J. Thermostable DNA polymerase chain amplification of    t(14;18) chromosome breakpoints and detection of minimal residual    disease. Proc Natl Acad Sci USA 1988; 85: 4869-4873.-   151. Lee M S. Molecular aspects of chromosomal translocation    t(14;18). Semin Hematol 1993; 30: 297-305.-   152. Buchonnet G, Lenain P, Ruminy P, Lepretre S, Stamatoullas A,    Parmentier F, Jardin F, Duval C, Tilly H, Bastard C.    Characterisation of BCL2-JH rearrangements in follicular lymphoma:    PCR detection of 3′ BCL2 breakpoints and evidence of a new cluster.    Leukemia 2000; 14: 1563-1569.-   153. Cleary M L, Galili N, Sklar J. Detection of a second t(14;18)    breakpoint cluster region in human follicular lymphomas. J Exp Med    1986; 164: 315-320.-   154. Akasaka T, Akasaka H, Yonetani N, Ohno H, Yamabe H, Fukuhara S,    Okuma M. Refinement of the BCL2/immunoglobulin heavy chain fusion    gene in t(14;18)(q32;q21) by polymerase chain reaction amplification    for long targets. Genes Chromosomes Cancer 1998; 21: 17-29.-   155. Willis T G, Jadayel D M, Coignet L J, Abdul-Rauf M, Treleaven J    G, Catovsky D, Dyer M J. Rapid. molecular cloning of rearrangements    of the IGHJ locus using long-distance inverse polymerase chain    reaction. Blood 1997; 90: 2456-2464.-   156. Yabumoto K, Akasaka T, Muramatsu M, Kadowaki N, Hayashi T, Ohno    H, Fukuhara S, Okuma M. Rearrangement of the 5′ cluster region of    the BCL2 gene in lymphoid neoplasm: a summary of nine cases.    Leukemia 1996; 10: 970-977.-   157. Pezzella F, Ralfkiaer E, Gatter K C, Mason D Y. The 14;18    translocation in European cases of follicular lymphoma: comparison    of Southern blotting and the polymerase chain reaction. Br J    Haematol 1990; 76: 58-64.-   158. Turner G E, Ross F M, Krajewski A S. Detection of t(14;18) in    British follicular lymphoma using cytogenetics, Southern blotting    and the polymerase chain reaction. Br J Haematol 1995; 89: 223-225.-   159. Vaandrager J W, Schuuring E, Raap T, Philippo K, Kleiverda K,    Kluin P. Interphase FISH detection of BCL2 rearrangement in    follicular lymphoma using breakpoint-flanking probes. Genes    Chromosomes Cancer 2000; 27: 85-94.-   160. Vaandrager J W, Schuuring E, Kluin-Nelemans H C, Dyer M J, Raap    A K, Kluin P M. DNA fiber fluorescence in situ hybridization    analysis of immunoglobulin class switching in B-cell neoplasia:    aberrant CH gene rearrangements in follicle center-cell lymphoma.    Blood 1998; 92: 2871-2878.-   161. Jacobson J O, Wilkes B M, Kwaiatkowski D J, Medeiros L J,    Aisenberg A C, Harris N L. bcl-2 rearrangements in de novo diffuse    large cell lymphoma. Association with distinctive clinical features.    Cancer 1993; 72: 231-236.-   162. Hill M E, MacLennan K A, Cunningham D C, Vaughan Hudson B,    Burke M, Clarke P, Di Stefano F, Anderson L, Vaughan Hudson G, Mason    D, Selby P, Linch D C. Prognostic significance of BCL-2 expression    and bcl-2 major breakpoint region rearrangement in diffuse large    cell non-Hodgkin's lymphoma: a British National Lymphoma    Investigation Study. Blood 1996; 88: 1046-1051.-   163. Vaandrager J W, Schuuring E, Philippo K, Kluin P M. V(D)J    recombinase-mediated transposition of the BCL2 gene to the IGH locus    in follicular lymphoma. Blood 2000; 96: 1947-1952.-   164. Fenton J A, Vaandrager J W, Aarts W M, Bende R J, Heering K,    van Dijk M, Morgan G, van Noesel C J, Schuuring E, Kluin P M.    Follicular lymphoma with a novel t(14;18) breakpoint involving the    immunoglobulin heavy chain switch mu region indicates an origin from    germinal center B cells. Blood 2002; 99: 716-718.-   165. Alaibac M, Filotico R, Giannella C, Paradiso A, Labriola A,    Marzullo F. The effect of fixation type on DNA extracted from    paraffin-embedded tissue for PCR studies in dermatopathology.    Dermatology 1997; 195: 105-107.-   166. An SF and Fleming K A. Removal of inhibitor(s) of the    polymerase chain reaction from formalin fixed, paraffin wax embedded    tissues. J Clin Pathol 1991; 44: 924-927.-   167. Camilleri-Broet S, Devez F, Tissier F, Ducruit V, Le Tourneau    A, Diebold J, Audouin J, Molina T. Quality control and sensitivity    of polymerase chain reaction techniques for the assessment of    immunoglobulin heavy chain gene rearrangements from fixed- and    paraffin-embedded samples. Ann Diagn Pathol 2000; 4: 71-76.-   168. Greer C E, Peterson S L, Kiviat N B, Manos M M. PCR    amplification from paraffin-embedded tissues. Effects of fixative    and fixation time. Am J Clin Pathol 1991; 95: 117-124.-   169. Legrand B, Mazancourt P, Durigon M, Khalifat V, Crainic K. DNA    genotyping of unbuffered formalin fixed paraffin embedded tissues.    Forensic Sci Int 2002; 125: 205-211.-   170. Lo Y M, Mehal W Z, Fleming K A. In vitro amplification of    hepatitis B virus sequences from liver tumour DNA and from paraffin    wax embedded tissues using the polymerase chain reaction. J Clin    Pathol 1989; 42: 840-846.-   171. Longy M, Duboue B, Soubeyran P, Moynet D. Method for the    purification of tissue DNA suitable for PCR after fixation with    Bouin's fluid. Uses and limitations in microsatellite typing. Diagn    Mol Pathol 1997; 6: 167-173.-   172. Sato Y, Sugie R, Tsuchiya B, Kameya T, Natori M, Mukai K.    Comparison of the DNA extraction methods for polymerase chain    reaction amplification from formalin-fixed and paraffin-embedded    tissues. Diagn Mol Pathol 2001; 10: 265-271.-   173. Tbakhi A, Totos G, Pettay J D, Myles J, Tubbs B R. The effect    of fixation on detection of B-cell clonality by polymerase chain    reaction. Mod Pathol 1999; 12: 272-278.-   174. Goelz S E, Hamilton S R, Vogelstein B. Purification of DNA from    formaldehyde fixed and paraffin embedded human tissue. Biochem    Biophys Res Commun 1985; 130: 118-126.-   175. Chan P K, Chan D P, To K F, Yu M Y, Cheung J L, Cheng A F.    Evaluation of extraction methods from paraffin wax embedded tissues    for PCR amplification of human and viral DNA. J Clin Pathol 2001;    54: 401-403.-   176. Coombs N J, Gough A C, Primrose J N. Optimisation of DNA and    RNA extraction from archival formalin-fixed tissue. Nucleic Acids    Res 1999; 27: e12.-   177. Wickham C L, Boyce M, Joyner M V, Sarsfield P, Wilkins B S,    Jones D B, Ellard S. Amplification of PCR products in excess of 600    base pairs using DNA extracted from decalcified, paraffin wax    embedded bone marrow trephine biopsies. Mol Pathol 2000; 53: 19-23.-   178. Cawkwell L and Quirke P. Direct multiplex amplification of DNA    from a formalin fixed, paraffin wax embedded tissue section. Mol    Pathol 2000; 53: 51-52.-   179. Diaz-Cano S J and Brady S P. DNA extraction from    formalin-fixed, paraffin-embedded tissues: protein digestion as a    limiting step for retrieval of high-quality DNA. Diagn Mol Pathol    1997; 6: 342-346.-   180. Hoeve M A, Krol A D, Philippo K, Derksen P W, Veenendaal R A,    Schuuring E, Kluin P M, van Krieken J H. Limitations of clonality    analysis of B cell proliferations using CDR3 polymerase chain    reaction. Mol Pathol 2000; 53: 194-200.-   181. Zhou X G, Sandvej K, Gregersen N, Hamilton-Dutoit S J.    Detection of clonal B cells in microdissected reactive    lymphoproliferations: possible diagnostic pitfalls in PCR analysis    of immunoglobulin heavy chain gene rearrangement. Mol Pathol 1999;    52: 104-110.

TABLE 1 B, T, and NK lineage of lymphoid rnalignancies^(a) Chroniclympho- Mul- ALL cytic Non-Hodgkin lymphomas tiple child- leuke- extra-mye- Lineage hood adult mias nodal nodal skin loma B 82-86% 75-80%95-97% 95-97% 90-95% 30-40% 100% T 14-18% 20-25% 3-5% 3-5%  5-10% 60-70% 0% NK <1% <1% 1-2% <2% <2% <2%  0% ^(a)See Van Dongen et al. 1991¹,Jaffe et at 2001 2, and Van Dongen et al. 2002 ⁵

TABLE 2 Estimated number of non-polymorphic human V, D, and J genesegments that can potentially be involved in Ig or TCR generearrangements^(a) Gene segment IGH IGK IGL TCRA TCRB TCRG TCRD Vsegments functional 44 (7)  43 (7) 38 (10) 46 (32) 47 (23) 6 (4) 8(family) 66 (7)^(b) 76 (7) 56 (11) 54 (32) 67 (30) 9 (4) 8 re- arrang-eable (family) D segments re- 27(7)  — — —  2 — 3 arrang- eable (family)J segments functional 6^(c) 5^(d) 4  53 13 5 4 re- 6^(c) 5^(d) 5^(e) 6113 5 4 arrang- eable ^(a)Only non-polymorphic gene segments with asuitable RSS are included in this table. ^(b)This estimation does notinclude the recently discovered (generally truncated) V_(H) pseudogenes,which are clustered in three clans ^(c)The six J_(H) gene segments arehighly homologous over a stretch of ~20 nucleotides, which is sufficientfor the design of a consensus primer. ^(d)The Jκ segments have a highhomology, which allows the design of 2 to 3 Jκ consensus primers.^(e)Five of the seven Jλ gene segments have a suitable RSS.

TABLE 3 Standardized PCR protocol Reaction conditions buffer: ABI BufferII or ABI Gold Buffer 50 μl final volume 100 ng DNA 10 pmol of eachprimer (unlabeled or 6-FAM labeled) (irrespective of total numbers ofprimers in each multiplex PCR tube) dNTP: 200 μM final concentrationMgCl₂: 1.5 mM final concentration (to be optimized per target) Tagenzyme^(a): 1U in most tubes; 2U in tubes with many primers (>15)Cycling conditions pre-activation 7 mm. at 95° C. annealing temperature:60° C. cycling times: “classical” “newer” PCR equipment PCR equipmentdenaturation 45 sec. 30 sec. annealing ≥45 sec. ≥30 sec. extension 1.30min. ≥30 sec. final extension ≥10 min. ≥10 min. number of cycles: 35hold 15° C. (or room temperature) ^(a)AmpliTaq Gold (Applied Biosystems,Foster City, CA) was used in combination with 1x ABI Buffer II or 1x ABIGold Buffer (Applied Biosystems), depending on the target.

TABLE 4 Standardized protocol for heteroduplex analysis of PCR productsPCR product preparation tube with 10-20 μl of PCR product denaturationof PCR product: 5 min. at 95° C. re-annealing of PCR product: 60 min. at4° C. Electrophoresis conditions (non-commercial polyacrylamide gels)gel: 6% non-denaturing polyacrylamide-(acrylamide: bisacrylamide 29:1)buffer: 0.5 × TBE loading buffer: 5 μl ice-cold non-denaturingbromophenol blue loading buffer electrophoresis: typically 2-3 hours at110 V or overnight at 40-50 V^(a) Electrophoresis conditions (commercialpolyacrylamide gels) gel: non-denaturing polyacrylamide (e.g. BioRadPreCast Gel System or Amersham Pharmacia Biotech Gene Gel Excel Kit)buffer: 1 × TBE loading buffer: ice-cold non-denaturing bromophenol blueloading buffer electrophoresis: 1.5 hours at 100 V Visualizationstaining: 5-10 min. in 0.5 μg/ml EtBr in H₂O destaining/washing: 2× 5-10mm. in H₂O visualization: UV illumination alternative: silver stainingusing Amersham Pharmacia Biotech DNA Silver stain kit ^(a)Voltage andelectrophoresis time depend on PCR amplicon sizes, thickness ofpolyacrylamide gel, and type of PCR equipment, and should be adaptedaccordingly.

TABLE 5 Standardized protocol for GeneScanning of PCR products A.Gel-based sequencers PCR product preparation 1. PCR product dilution:initially 1:10 in formamide or H₂O (can be altered if fluorescent signalis outside optimal range; see electrophoresis conditions) 2. samplevolume: 2 μl diluted PCR product 3. loading buffer volume: 0.5 μl bluedextran loading buffer + 0.5 μl TAMRA internal standard + 2 μl deionizedformamide 4. denaturation of PCR product: 2 min at 95° C. or highertemperature 5. cooling of PCR product at 4° C. Electrophoresisconditions 6. gel: 5% denaturing polyacrylamide 7. buffer: 1 × TBE 8.electrophoresis: 2-3.5 hours^(a) (see Table 25) 9. optimal fluorescentsignal intensity: 600-4,000 fluorescent units (373 platforms) 400-7,000fluorescent units (377 platforms) B. Capillary sequencers (to beoptimized per sequencer) PCR product preparation 1. 1 μl PCR product(volume of PCR product or sampling times can be altered if fluorescentsignal is outside optimal range; see electrophoresis conditions) 2.sample volume: 1 μl PCR product + 9.5 μl (Hi-Di) formamide + 0.5 μlROX-400 heteroduplex analysis internal standard 3. denaturation of PCRproduct: 2 min. at 95° C. or higher temperature 4. cooling of PCRproduct at 4° C. for an hour Electrophoresis conditions 5. gel: 3100POP4 polymer 6. buffer: 1× 3100 buffer with EDTA 7. electrophoresis: 45minutes^(b) 8. optimal fluorescent signal intensity: up to 10,000fluorescent units ^(a)Electrophoresis time depends on amplicon sizes andon employed platform. ^(b)For 36 cm capillary; time taken depends oncapillary used.

TABLE 6 Sensitivity of detection of clonal TCRB rearrangementsSensitivity Involved of detection primer Size of multi- TCRB pair ClonalPCR single plex tube V J Control product PCR^(a) PCR tube A Vβ2 Jβ1.2patient 261 nt   1-5%  5% Vβ2 Jβ1.3 patient 267 nt   5%  5% Vβ2 Jβ1.6patient 267 nt  <5%   Vβ7 Jβ2.2 patient 254 nt 10%  Vβ8a Jβ1.2 Jurkat267 nt 0.1% 0.5-1%  Vβ8a Jβ2.7 patient 264 nt 10%  Vβ10 Jβ2.7 PEER 263nt 20% Vβ3/12a/ Jβ1.6 patient 278 nt <5%  5% 13a/15 Vβ3/12a/ Jβ2.7patient 286 nt 10% 13a/15  Vβ17 Jβ2.7 RPMI- 260 nt 10% 8402  Vβ17 Jβ1.1patient 260 nt   1% 10%  Vβ18 Jβ1.2 DND41 261 nt   1% 10%  Vβ22 Jβ1.1patient 265 nt 0.1% 10% Vβ8b/23 Jβ1.2 E19 257 nt 0.1% 0.5%   Vβ24 Jβ1.5RPMI- 264 nt 0.5% 10% 8402 tube B Vβ2 Jβ2.1 Molt-4 267 nt   5%  5%  Vβ1/5 Jβ2.1 patient 266 nt   5% 1-5%  Vβ6a/11 Jβ2.1 patient 265 nt  1%  5% Vβ6a/11 Jβ2.5 patient 258 nt  5% Vβ7 Jβ2.3 PEER 271 nt  <5%   Vβ8a Jβ2.1 patient 293 nt 0.1%  1% Vβ3/12a/ 13a/15 Jβ2.1 patient 258 nt  5% 10% Vβ3/12a/ 13a/15 Jβ2.3 patient 258 nt  <5%    Vβ16 Jβ2.1 patient258 nt 0.5% 10%  Vβ17 Jβ2.5 CML-T1 270 nt 0.1-1%  1%  Vβ21 Jβ2.3 patient282 nt 0.5% <10%   tube C Dβ1 Jβ1.1 patient 304 nt 0.10%   <5%   Dβ1Jβ1.2 patient 306 nt   5%  5% Dβ1 Jβ1.4 patient 310 nt 5-10% Dβ1 Jβ1.6patient 320 nt 20% Dβ1 Jβ2.1 patient 309 nt   5% 20% Dβ1 Jβ2.7 patient307 nt  <5%   Dβ1 Jβ2.5 patient 310 nt  <1%   Dβ2 Jβ1.4 patient 182 nt <1%   Dβ2 Jβ2.1 patient 185 nt   1%  <5%   Dβ2 Jβ2.5 patient 191 nt  5%^(a)The dilution experiment for assessing the sensitivity of the singlePCR was not performed in each case.

TABLE 7 Sensitivity of detection of clonal TCRD gene rearrangementsClonal control Sensitivity of TCRD sample detection by rearrangement(approximate size) heteroduplex Vδ1-Jδ1 patient (200 nt) 5% patient (190nt) 1-5%   patient (200 nt) 5% Vδ2-Jδ1 patient (200 nt) 5% patient (220nt) 5% patient (210 nt) 5% Vδ2-Jδ3 patient (220 nt) 5% Vδ3-Jδ1 patient(270 nt) 5% Vδ6-Jδ2 Loucy (210 nt) 0.5%   patient (210 nt) 10%  Dδ2-Jδ1Loucy (150 nt) 0.2%   patient (160 nt) 0.5%   patient (135 nt) 0.5%  Dδ2-Jδ3 patient (150 nt) 5% Dδ2-Dδ3 NALM-16 (170 nt) 1% patient (200 nt)1% patient (190 nt) 0.5%   patient (170 nt) 0.5%   Vδ2-Dδ3 REH (240 nt)5-10%   NALM-16 (230 nt) 1-5%   patient (250 nt) 5%

TABLE 8 Concordance between multiplex PCR results and Southern blot (SB)analysis results (PCR/SB) on Ig/TCR gene rearrangements per(sub)category of included frozen samples Diagnosis IGH^(a) IGK IGL TCRBTCRG TCRD pre-follicular C^(b): 8/8 C: 8/8 C: 4/4 C: 2/4^(b) C: 0/0 C:0/0 (n = 8) P^(b): 0/0 P: 0/0 P: 4/4 P: 4/4 P: 8/8 P: 8/8^(e) B-CLL (n =16) C: 15/16 C: 16/16 C: 5/5 C: 1/1 C: 0/0 C: 2/2 P: 0/0 P: 0/0 P: 9/11P: 15/15 P: 16/16 P: 14/14 (post-)follicular C: 22/25^(b) C: 19/24^(c)C: 3/5 C: 2/4 C: 0/1 C: 0/0 (n = 25) P: 0/0 P: 0/1 P: 19/20 P:21/21^(d,e) P: 22/24 P: 24/25^(e) All B-cell C: 45/49 C: 43/48 C: 12/14C: 4/8 C: 0/1 C: 2/2 malignancies P: 0/0 P: 0/1 P: 32/35 P: 41/41 P:46/48 P: 46/47 (n = 49) T-cell C: 2/2 C: 0/0 C: 0/0 C: 17/17c C:15/16^(b) C: 2/3 malignancies P: 15/16^(e) P: 17/18 P: 17/18 P: 1/1 P:1/2 P: 14/159 (n = 18) Reactive samples C: 0/0 C: 0/0 C: 0/0 C: 0/0 C:0/0 C: 0/0 (n = 15) P: 15/15 P: 15/15 P: 15/15 P: 14/15 P: 15/15 P:15/15 Miscellaneous C: 3/3 C: 2/2 C: 0/0 C: 3/3 C: 1/1 C: 1/1 (n = 8) P:3/5 P: 4/6 P: 6/8 P: 5/5^(d,d) P: 6/7 P: 5/7 All samples C: 50/54 C:45/50 C: 12/14 C: 25/29 C: 16/18 C: 5/6 (n = 90) P: 33/36 P: 36/40 P:70/76 P: 60/61 P: 68/72 P: 80/84 ^(a)Includes both VH-JH and DH-JH PCRanalysis ^(b)C, clonal rearrangements; P, polyclonal rearrangements^(c)In one sample clonality in GeneScanning only ^(d)In one sampleclonality in heteroduplex analysis only ^(e)In one sample polyclonalityin GeneScanning only ^(f)In one sample polyclonality in heteroduplexanalysis only

TABLE 9 Complementarity of different Ig multiplex PCR targets forclonality detection in Southern blot-defined B-cell malignanciesDiagnosis^(a) Multi- Pre- (post-) all B-cell plex germinal germinalmalig- PCR center B B-CLL center B nancies tubes (n = 8) (n = 16) (n =25) (n = 49) IGH 8/8^(b) (100%) 14/16^(c) (88%) 15/25^(b) (60%) 37/49(76%) VH- JH FR1 IGH 8/8 (100%) 15/16 (94%) 14/25^(b) (56%) 37/49 (76%)VH- JH FR2 IGH 8/8 (100%) 14/16 (88%) 11/25^(c) (44%) 33/49 (67%) VH- JHFR3 IGH 8/8 (100%) 15/16 (94%) 17/25 (68%) 40/49 (82%) VH- JH 3FR IGH0/8 (0%) 11/16 (69%) 11/25 (44%) 22/49 (45%) D_(H)-J_(H) IGH 8/8 (100%)15/16 (94%) 22/25 (88%) 45/49 (92%) VH- JH + IGH DH-JH IGK 8/8 (100%)16/16 (100%) 21/25^(d) (84%) 45/49 (92%) IGL 4/8 (50%) 7/16^(a) (44%)4/25^(f) (16%) 15/49 (31%) IGH 8/8 (100%) 16/16 (100%) 21/25 (84%) 45/49(92%) VH- JH + IGK IGH 8/8 (100%) 15/16 (94%) 17/25 (68%) 40/49 (82%)VH- JH + IGL IGH 8/8 (100%) 16/16 (100%) 24/25 (96%) 48/49 (98%) VH-JH + IGH DH- JH + IGK IGH 8/8 (100%) 16/16 (100%) 24/25 (96%) 48/49(98%) VH- JH + IGH DH- JH + IGK + IGL ^(a)All samples have clonal generearrangements in at least the IGH locus as determined by Southern blotanalysis ^(b)Two cases showed clonal products in GeneScanning, butpolyclonal products in heteroduplex analysis ^(c)One case showed clonalproducts in GeneScanning, but polyclonal products in heteroduplexanalysis ^(d)Including case 25-NL-4 with weak clonal IGH but polyclonalIGK gene rearrangements in Southern blot analysis ^(e)Including cases11-NL-19 and 12-ES-1 with clonal IGH + IGK but polyclonal IGL generearrangements in Southern blot analysis

TABLE 10 Conditions and control samples for multiplex PCR analysis ofIg/TCR gene rearrangements and translocations t(11; 14) and t(14; 18)Positive Multi- PCR conditions controls (examples) plex TaqGold MgCl₂poly- PCR Tubes Buffer (U) (mM) clonal monoclonal^(a) IGH A/B/C Gold/II1 1.5 tonsil A: NALM-6; VH- SU-DHL-5; JH SU-DHL-6 B: NALM-6; SU-DHL-5;SU-DHL-6 C: NALM-6; SU-DHL-5; SU-DHL-6 IGH D/E Gold 1 1.5 tonsil D: KCA;ROS15 DH- E: HSB-2, HPB- JH ALL IGK A/B Gold/II 1 1.5 tonsil A: KCA;R0S15 B: ROS15, 380 IGL A Gold/II 1 2.5 tonsil A: CLL-1; EB-4B; KCA TCRBA/B/C II 2 3.0 PB- A: RPMI-8402; (A, B)^(b) (A, B) MNC^(c) Jurkat; PEER;1 (C) 1.5 (C) DND-41 B: PEER; CML- T1, MOLT-3 C: Jurkat TCRG A/B II 11.5 PB- A: MOLT-3; MNC^(c) RPMI-8402; Jurkat; PEER B: Jurkat; PEER TCRDA II 1 2.0 PB- A: PEER, REH MNC^(c) BCL1- A II 1 2.0 NAC A: JVM 2 IGHBCL2- A/B/C II 1 1.5 NAC A: DoHH2; SU- IGH DHL-6 B: K231^(d) C: OZ;SC1^(d); SU- DHL-16 ^(a)Most clonal cell line controls can be obtainedvia the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH;contact person: dr. H. G. Drexler (address: Department of Human andAnimal Cell Cultures, Mascheroder Weg 1B, 38124 Braunschweig, Germany).^(192, 193) ^(b)In most multiplex tubes only 1 U TaqGold is needed, but2 U TaqGold are needed in TCRB tubes A and B because they contain >15different primers. ^(c)Abbreviations: PB-MNC, mononuclear cells fromperipheral blood; NA, not applicable. ^(d)The t(14; 18) positive celllines K231, OZ, and SC1 were kindly provided by prof. Martin Dyer,University of Leicester, Leicester, GB.

TABLE 11 Size ranges, non-specific bands, and detection method inmultiplex PCR analysis of Ig/TCR gene rearrangements and chromosomeaberrations t(11; 14) and t(14; 8) GeneScan Multi- Preferred runningplex Nonspecific method of time: gel/ PCR Size range (bp) bands (bp)analysis capillary IGH Tube A: 310-360 Tube A: ~85 GeneScanning 3-3.5 h/VH-JH Tube B: 250-295 Tube B: — and heteroduplex 45 min Tube C: 100-170Tube C: — analysis equally suitable IGH Tube D: 110-290 Tube D: 350^(a)heteroduplex 3-3.5 h/ DH-JH (D_(H)1/2/4/5/6- Tube E: 211^(b) analysisslightly 45 min J_(H) + 390-420 preferred over (D_(H)3-J_(H))GeneScanning Tube E: 100-130 (variation of product sizes hampersGeneScanning) IGK Tube A: 120-160 Tube A: — heteroduplex 3-3.5 h/(Vκ1f/6/Vκ7- Tube B: ~404 analysis slightly 45 min Jκ) + 190-210preferred over (Vκ3f-Jκ) + GeneScanning 260-300 (Vκ2f/ (small junctionVκ4/Vκ5-Jκ) size + variation Tube B: 210-250 of product sizesVκ1f/6/Vκ7- hampers κde + 270-300 GeneScanning) (W3f/intron- Kde) +350-390 (Vκ2f/Vκ4/ Vκ5-Kde) IGL Tube A: 140-165 Tube A: — heteroduplex 2h/ analysis clearly 45 min preferred over GeneScanning (small junctionsize hampers GeneScanning) TCRB Tube A: 240-285 Tube A: (273)^(c)heteroduplex 2 h/ Tube B: 240-285 Tube B: <150, 221^(d) analysisslightly 45 min Tube C: 170-210 (Dβ2) + Tube C: 128, 337^(d) preferredover 285-325 (Dβ1) GeneScanning (limited repertoire, particularly incase of ψVγ10 and kψVγ11 usage) TCRG Tube A: 145-255 Tube A: —GeneScanning 2 h/ Tube B: 80-220 Tube B: — and 45 min heteroduplexanalysis equally suitable TCRD Tube A: 120-280 Tube A: ~90 heteroduplex2 h/ analysis clearly 45 min preferred over GeneScanning (low amount oftemplate + variation of product sizes hampers GeneScanning) BCL1- TubeA: 150-2000 Tube A: ~550 agarose NA^(e) IGH (weak) BCL2- Tube A:variable Tube A: — agarose NA^(e) IGH Tube B: variable Tube B: — Tube C:variable Tube C: — ^(a)The nonspecific 350 bp band is the result ofcross-annealing of the D_(H)2 primer to a sequence in the regionupstream of J_(H)4. In GeneScanning this nonspecific band does notcomigrate with D-J products (see FIG. 5B). ^(b)The 211 bp PCR productrepresents the smallest background band derived from the germlineD_(H)7-J_(H)1 region. When the PCR amplification is very efficient, alsolonger PCR products might be obtained because of primer annealing todownstream J_(H) gene rearrangements; e.g. 419 bp (D_(H)7-J_(H)2), 1031bp (D_(H)7-J_(H)3), etc. ^(c)The 273 bp band (mainly visible byGeneScanning) is particularly seen in samples with low numbers ofcontaminating lymphoid cells. ^(d)Intensity of non-specific band dependson primer quality. ^(e)NA, not applicable

1. A set of nucleic amplification primers capable of amplifying aV_(H)-J_(H) IGH rearrangement comprising a forward primer and a reverseprimer, wherein said forward primer is selected from the V_(H) familyprimers shown in FIG. 3B, or a variant thereof, and wherein said reverseprimer is the J_(H) consensus primer shown in FIG. 3B, or a variantthereof. 2-7. (canceled)
 8. A set of nucleic amplification primerscapable of amplifying a Vγ-Jγ TCRG rearrangement comprising a forwardprimer and a reverse primer, wherein said forward primer is selectedfrom the Vγ family primers shown in FIG. 8B, or a variant thereof, andwherein said reverse primer is selected from the Jγ primers shown inFIG. 8B, or a variant thereof. 9-13. (canceled)
 14. A set of nucleicamplification primers capable of amplifying a chromosomal translocationt(14;18)(BCL2-IGH), comprising a forward primer and a reverse primer,wherein said forward primer is selected from the MBR primers, the 3′MBRprimers and the mer primers shown in FIG. 11A, or a variant thereof, andwherein said reverse primer is the J_(H) consensus primer shown in FIG.11A, or a variant thereof. 15-19. (canceled)
 20. A nucleic acidamplification assay, preferably a PCR assay, more preferably a multiplexPCR assay, using at least one set of primers according to claim 14.21-27. (canceled)
 28. A method for detecting a Vγ-Jγ TCRG rearrangement,comprising using one or more sets of primers according to claim 8 in anucleic acid amplification assay, preferably a PCR assay, morepreferably a multiplex PCR assay. 29-33. (canceled)
 34. A method fordetecting a chromosomal translocation t(14;18)(BCL2-IGH), comprisingusing one or more sets of primers according to claim 14 in a nucleicacid amplification assay, preferably a PCR assay, more preferably amultiplex PCR assay. 35-42. (canceled)
 43. A nucleic acid amplificationassay, preferably a PCR assay, more preferably a multiplex PCR assay,using at least one set of primers according to claim 8.